Apparatus, methods and composition for synthesis of cannabinoid compounds

ABSTRACT

The disclosure provides systems and methods for producing a cannabinoid product, which comprises contacting a cannabinoid precursor in a first phase with a cannabinoid synthase in a second phase, wherein the first phase and the second phase are substantially immiscible or immiscible. The disclosure also provides a composition comprising the cannabinoid precursor in a first phase and a cannabinoid synthase in a second phase, wherein the first phase and the second phase are substantially immiscible or immiscible.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/976,487, filed May 10, 2018, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Nos. 62/490,577, filed Apr. 26,2017, 62/490,579, filed on Apr. 26, 2017, 62/514,617, filed on Jun. 2,2017, and 62/514,626, filed on Jun. 2, 2017. The content of eachapplication is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Cannabinoids are terpenophenolic compounds found in Cannabis sativa, anannual plant belonging to the Cannabaceae family. The plant producesmore than 100 different cannabinoids. Cannabinoids accumulate mainly inthe glandular trichomes. Classical cannabinoid compounds includetetrahydrocannabinol (THC), prescribed by physicians as dronabinol(Marinol®) or nabilone (Cesamet®), which is used for treating glaucoma,AIDS wasting, and chemotherapy-induced nausea. THC may also be effectivein the treatment of allergies, inflammation, epilepsy, depression,migraine, bipolar disorders, anxiety disorder, drug dependency,neuropathic pain, treatment of spasticity associated with multiplesclerosis, fibromyalgia, and drug withdrawal syndromes.

Cannabinoids have therapeutic potential. For example, cannabidiol (CBD)is a potent antioxidant and anti-inflammatory compound and may provideprotection against acute and chronic neuro-degeneration. It is found inhigh concentrations in hemp and acts as a high affinity α2-adrenergicreceptor agonist, moderate affinity 5-HT1A receptor antagonist and lowaffinity CB1 receptor antagonist. CBD may also have anti-depressantactivity. Cannabichromene (CBC) possesses anti-inflammatory,anti-fungal, and anti-viral properties. Thus, cannabinoids areconsidered to be promising agents for their beneficial effects in thetreatment of various diseases.

The varins are a class of cannabinoids that are structurally differentfrom the classical cannabinoids (e.g., THC, CBD, CBG, or CBC). Insteadof having a pentyl (5-carbon) side chain attached to the aromatic ringas present in the classical cannabinoids, varins have a 3-carbon propylside chain. Many of the varins are found in very low amounts in theCannabis plant. Tetrahydrocannabivarin (THCV) is one of the most studiedcannabinoid varin compounds. THCV can function as an antagonist of THCat CB1 receptors and thus attenuate the psychoactive effects of THC.THCV has also been shown as a potential treatment for type 2 diabetes byincreasing insulin sensitivities and improving glucose tolerance.Wargent et al., Nutr Diabetes., May; 3(5): e68 (2013). THCV has alsoshown promise for treatment of epilepsy and to reduce tremors associatedwith Parkinson's diseases.

Despite their known beneficial effects, therapeutic use of cannabinoidcompounds, particularly varins, is hampered by the difficulty inobtaining high yields of cannabinoid compounds (both pentyl and propylchain cannabinoids) from plants. Moreover, extraction, isolation, andpurification of cannabinoid compounds from plant tissue are particularlychallenging for a variety of reasons, including the difficulty ofseparating cannabinoids from terpenes, chlorophyll, and other plantcomponents and the fact that the Cannabis plant only produces smallquantities of many of these cannabinoids.

Therefore, the practical challenges in isolating the natural cannabinoidcompounds from plants highlights a need for developing effective, safesystems or methods for large scale production of cannabinoid compoundsfor therapeutic use, especially, since chemical methods for synthesizingmany of the cannabinoids and rarer varins are not yet available in thepublished literature.

SUMMARY OF INVENTION

It is therefore an object of the disclosure to provide solutions to theaforementioned deficiencies in the art. To this end, the presentdisclosure relates generally to systems and methods for producing acannabinoid product. In one embodiment, the system for producing acannabinoid or its analog, comprising: a) fermentor holding a medium anda plurality of cells, wherein the cells are configured to produce andsecrete cannabinoid synthase; b) a bioreactor containing a cannabinoidprecursor in a first phase with a cannabinoid synthase in a secondphase, c) a control mechanism configured to control a condition of thebioreactor, wherein the condition of the bioreactor influences aquantity formed of the first cannabinoid relative to a quantity formedof a second cannabinoid or a cannabinoid analog. In another embodiment,the method comprises contacting a cannabinoid precursor in a first phasewith a cannabinoid synthase in a second phase. The first phase and thesecond phase, in some embodiments, are substantially immiscible orimmiscible. In one embodiment, the cannabinoid synthase used in thisdisclosure comprises cannabidiolic acid (CBDA) synthase,tetrahydrocannabinolic acid (THCA) synthase, cannabichromenic acid(CBCA) synthase, or a combination thereof. With regard to THCA synthaseand CBDA synthase, the cannabinoid precursor is a compound according toFormula I:

wherein R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b) group;R₁ is —H, —COOH, or —C(O)R_(a), and R₂ is selected from the groupconsisting of —H, —C(O)R_(a), —OR_(a), an optionally substituted C₁-C₁₀linear or branched alkylene, an optionally substituted C₂-C₁₀ linear orbranched alkenylene, an optionally substituted C₂-C₁₀ linear or branchedalkynylene, an optionally substituted C₃-C₁₀ aryl, an optionallysubstituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene. Inone embodiment, R₁ and R₂ together with the carbon atoms to which theyare bonded form a C₅-C₁₀ cyclic ring; R₃ is selected from the groupconsisting of H, —C(O)R_(a), and C₁-C₁₀ linear or branched alkyl; andR_(a) and R_(b) are each independently —H, —OH, —SH, —NH₂, (C₁-C₁₀)linear or branched alkyl, or a C₃-C₁₀ cycloalkyl. In another embodiment,the cannabinoid precursor comprises cannabigerolic acid (CBGA),cannabigerovaniric acid (CBGVA), or the combination thereof.

The disclosure also relates to compositions, which can be used for, butare not limited to, synthesizing the cannabinoids. The compositionscomprise (a) a cannabinoid precursor in a first phase; and (b) acannabinoid synthase enzyme in a second phase, wherein the first phaseand the second phase are substantially immiscible or immiscible. In someembodiments, the first phase comprises an organic solvent that iswater-immiscible or substantially water-immiscible, and the second phasecomprises an aqueous solvent or a mixture of an aqueous and a miscibleorganic solvent. In one embodiment, the cannabinoid synthase used inthis disclosure comprises cannabidiolic acid (CBDA) synthase,tetrahydrocannabinolic acid (THCA) synthase, cannabichromenic acid(CBCA) synthase, or combination thereof. The cannabinoid precursor is acompound according to Formula I.

Also provided is an apparatus for the ex vivo manufacture ofcannabinoids and analogs of cannabinoids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an apparatus for synthesis of cannabinoids usingbiphasic production system and its communication mechanism.

FIG. 2 shows chemical structures of cannabinoid compounds, includingTHCVA, CBDVA, CBCVA, THCV, CBCV, CBDV, THCA, CBDA, CBCA, THC, CBD, CBC,CBNA, CBCLA, CDB difluoromethyl ether, and CBD methyl ether.

FIGS. 3A-3B shows the aqueous solutions with CBGVA (FIG. 3A) and CBGA(FIG. 3B) in aqueous buffer. At 0.05 g/L concentration, precipitationwas observed in the CBGA solution (FIG. 3B), but not in the CBGVAsolution.

FIG. 4 shows crystal formation after 24 hours in 0.1 g/L CBGVA solutionin pH4.5 citrate buffer and 5% DMSO under phase contrast microscope with400× magnification.

FIGS. 5A-5B shows the results of cannabinoid synthesis in a 1:1 biphasicoil-aqueous reaction with 32 mg/mL lyophilized cannabinoid synthases(THCA synthase for FIG. 5A and CBDA synthase for FIG. 5B). In thebiphasic system, the pH of the aqueous phase is 5.5 with 10% DMSO, andthe oil phase solution contains 5 g/L CBGVA.

FIGS. 6A-6B depicts the activity of purified CBDA synthase in a 1:1biphasic oil-aqueous reaction with CBGA as substrate.

FIGS. 7A-7B and 8A-8B show effects of pH values on production ofcannabinoids in 1:1 biphasic soybean oil-aqueous reactions withlyophilized THCA synthase. The aqueous phase contains 32 mg/mLlyophilized THCA synthase. FIG. 7 shows the production of THCVA, whileFIG. 8 shows the production of CBCVA. Ratios of THCVA:CBVCA at 168 hoursare shown at FIG. 7B, and ratios of CBCVA:THCVA are shown in FIG. 8B.

FIG. 9 shows the standard curve for quantifying CBGVA in solution.

FIGS. 10A-10B and 11A-11B show effects of pH values on production ofcannabinoids in 1:1 biphasic soybean oil-aqueous reactions withlyophilized CBDA synthase. The aqueous phase contains 32 mg/mLlyophilized CBDA synthase. FIGS. 10A-10B show the production of CBDVA,while FIGS. 11A-11B show the production of CBCVA. Ratios of CBDVA:CBCVAare shown in FIG. 10B, and ratios of CBCVA:CBDA are shown in FIG. 11B.

FIGS. 12A-12B, 13A-13B, 14A-14B, 15A-15B show effects of DMSO on CBGVAcyclization by THCA and CBDA synthases in a biphasic oil-aqueous system.

FIGS. 16 and 17 depict the activity of THCA synthase in biphasicoil-aqueous reactions with CBGVA as substrate. In those experiments, pHwas optimized for formation of THCVA (FIG. 16) and CBCVA (FIG. 17),separately.

FIG. 18 depicts the activity of CBDA synthase in a biphasic oil-aqueousreaction with CBGVA as substrate.

FIG. 19 shows the chromatographic identifications of varin series ofcompounds by RP-HPLC.

FIGS. 20A-20B show the results of cannabinoid synthesis in a 1:1biphasic oil-aqueous reaction with 32 mg/mL lyophilized cannabinoidsynthases (THCA synthase for FIG. 20A and CBDA synthase for FIG. 20B).In the biphasic system, the aqueous phase solution was at pH 5.5 with10% DMSO, and the oil phase solution contains 5 g/L CBGA in oil phase.

FIGS. 21A-21B depict the activity of purified CBDA synthase in a 1:1biphasic oil-aqueous reaction with CBGA as substrate.

FIGS. 22A-22B and 23A-23B show effects of pH values on production ofcannabinoids in 2:1 biphasic soybean oil-aqueous reactions withlyophilized THCA synthase. The aqueous phase contains 32 mg/mLlyophilized THCA synthase. FIG. 22 shows the production of THCA, whileFIG. 23 shows the production of CBCA. Ratios of THCA:CBCA at 168 hoursare shown at FIG. 22B, and ratios of CBCA:THCA are shown in FIG. 23B.

FIG. 24 shows the production of THCA and CBCA in biphasic oil-aqueoussystems with different oil to aqueous ratios. All systems contain 20 mgof CBGA in soybean oil phase and 40 mg of Lyophilized Enzyme in 100 mMsodium citrate and 20% DMSO at pH 5.5.

FIGS. 25A-25B shows ratios of reaction products in biphasic oil-aqueoussystems with different oil:aqueous ratios. Ratios of products at eachtime point are shown in FIG. 25A. Ratios of products at 408 hours areshown in FIG. 25B.

FIG. 26 shows percentage of each cannabinoid by HPLC Area % (AUC) foreach oil:aqueous ratio when extracting just the oil layer vs. the total(oil and aqueous) assay with IPA.

FIG. 27 shows the total amount of each cannabinoid (mg) for eachoil:aqueous ratio when extracting just the oil layer compared to thetotal (oil and aqueous) assay with IPA.

FIGS. 28A-28B and 29 show production of cannabinoids in 1:1 biphasicoil-aqueous reactions with purified THCA Synthase at different pH (pH5.5 for FIG. 28 and pH 7.5 for FIG. 29) and different concentration ofDMSO.

FIGS. 30 and 31A-31B show the activities of purified THCA synthase inbiphasic oil-aqueous systems (1:1) with lower DMSO concentrations. FIG.30 shows the total amount of cannabinoids produced. FIG. 32 shows theratios of THCA to CBCA.

FIGS. 32, 33A-33B, 34A-34B, 35A-35B show the kinetics of CBGAcyclization in the presence of different concentrations of methanol andDMSO in an aqueous reaction system. FIG. 32 shows the UV-HPLC trace(detection at 267 nm) of products in the reaction of CBGA cyclizationcatalyzed by CBDA synthase in the presence of 10% (v/v) MeOH after 2hours (reaction products are marked by arrows). FIGS. 33A-33B showkinetics of CBGA conversion into CBDA (in relative units). FIGS. 34A-34Bshow kinetics of CBGA conversion into THCA (in relative units). FIG.35A-35B show kinetics of CBGA conversion into CBCA (in relative units).

FIG. 36 shows the THCA synthase activity after lyophilization.

FIGS. 37A-37B shows stability of CBDA synthase (20 mg/mL) afterincubation in 0.1 M citrate buffer, pH 4.5, in the presence of differentconcentrations of polar co-solvents.

FIGS. 38 and 39 depict the activities of THCA synthase in biphasicoil-aqueous reactions with CBGA as substrate. In those experiments, pHwas optimized for formation of CBCA (FIG. 38) and THCA (FIG. 39),separately.

FIG. 40 depicts the activity of CBDA synthase in a biphasic oil-aqueousreaction with CBGA as substrate.

FIG. 41 shows kinetics of conversion of CBGVA to THCVA and CBCVA in the3 L reaction.

FIG. 42 shows the amount of cannabinoids over the course of the reactionestimated using HPLC standard curves.

FIG. 43 shows the estimated amount of THCVA present in original oilemulsion and the various extraction stages.

FIG. 44 shows the elution profiles of cannabinoids from small-scalesilica column.

FIG. 45 shows conversion of CBGVA to THCVA and CBCVA over the first 55hours of reaction.

FIG. 46 shows the percentage of THCVA (by HPLC peak area) in reactionswith recycled enzyme.

FIGS. 47A-47B show recycle of aqueous phase using dipentene as theorganic phase and tech-grade enzyme (A) or enriched enzyme (B).

FIG. 48A-48B show recycle of aqueous phase using soybean oil as theorganic phase and tech-grade enzyme (A) or enriched enzyme (B).

FIG. 49 shows the overall THCVA production of each recycle reaction.

FIGS. 50A-50F shows the bioconversion of CBGVA with THCA synthase inpresence of alternative organic solvent. FIG. 50A shows the CBGVAconcentration during the bioconversion reaction at variousconcentrations of THCA synthase in presence of dipentene and catalase.FIG. 50B shows the CBGVA concentration during the bioconversion reactionat various concentrations of DMSO in presence of catalase. FIG. 50Cshows the concentrations of various cannabinoids after 48 hours ofreaction at various concentrations of DMSO. FIG. 50D shows the scale-upbioconversion reaction (300 mL) with 3 g of CBGVA in the 100 mLdipentene organic phase (30 g/L). The aqueous phase contains 200 mL ofpH 5.0 sodium citrate buffer, 10% DMSO with 5 g of THCA synthase (25g/L), and 20 mg of catalase (0.1 g/L). FIG. 50E depicts CBCVA productionin biphasic systems using different solvents and cosolvents. FIG. 50Fshows the ratio of CBCVA:THCVA at 20 hours of reactions time in presenceof dipentene along with DMSO and methanol.

FIGS. 51A-51F shows the bioconversion of CBGVA with CBDA synthase inpresence of alternative organic solvent. FIG. 51A depicts the conversionto CBDVA in presence of dipentene and soybean oil. FIG. 51B shows theconversion to CBDVA in dipentene in presence of catalase or MeOHcosolvent. FIG. 51C shows the conversion to CBDVA in soybean oil inpresence of catalase or MeOH cosolvent. FIG. 51D shows the conversion ofCBGVA to the whole cannabinoid products (CBDVA, THCVA, and CBCVA) indipentene with catalase or MeOH cosolvent. FIGS. 51E and 51F show thetotal cannabinoid production (CBDVA, THCVA, & CBCVA) in area percentover 144 hours in both biphasic soybean systems (FIG. 51E) and dipentenesystems (FIG. 51F).

FIG. 52 shows comparison of substrate concentrations in the aqueousphase for the dipentene and soybean oil reactions.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,compounds, polymers, and reagents described, as such may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tolimit the scope of the present invention, which will be limited only bythe appended claims.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “acompound” includes a plurality of compounds, and a reference to “amolecule” is a reference to one or more molecules.

All numerical designations, e.g., pH, temperature, time, concentration,amounts, and molecular weight, including ranges, are approximationswhich are varied (+) or (−) by 10%, 1%, or 0.1%, as appropriate. It isto be understood, although not always explicitly stated, that allnumerical designations may be preceded by the term “about.” It is alsoto be understood, although not always explicitly stated, that thereagents described herein are merely exemplary and that equivalents ofsuch are known in the art.

The term “comprising” or “comprises” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of,” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. For example, a compositionconsisting essentially of the elements as defined herein would notexclude other elements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than a trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

The term “co-solvent” is used to mean a solvent that is added to thefirst phase or the second phase in an amount less than 50% of the totalvolume. In one embodiment, the co-solvent in the first phase is awater-immiscible solvent. In another embodiment, the co-solvent in thesecond phase is a water-miscible solvent.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only, or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending uponthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art, given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

As used herein, the term “precursor” refers to a compound thatparticipates in a chemical reaction that produces another compound. Inone embodiment, the cannabinoid precursor refers to a compound thatparticipates in a reaction to produce another compound. For examples,CBGA is a precursor to THCA, CBDA, and CBCA. In another example, CBGVAis a precursor to THCVA, CBDVA, and CBCVA.

The term “cannabinoid product” or “cannabinoid compound” is intended tomean any simple or complex substance or compound of natural,semi-synthetic, or synthetic origin, which can act on the cannabinoidreceptors of a subject. In some embodiments, the cannabinoid product isan agonist of the cannabinoid receptor. In some embodiments, thecannabinoid product is an antagonist of the cannabinoid receptor. In oneembodiment, the cannabinoid product comprises phytocannabinoids,endogenous cannabinoids (endocannabinoids), bio-synthetic cannabinoids,or synthetic cannabinoids produced in laboratories. In one embodiment,the cannabinoid product comprises a pentyl side chain on the aromaticring. Certain cannabinoids have a propyl side chain. In thisapplication, this class of cannabinoids may be referred to as “varin.”

Non-limiting cannabinoid products include tetrahydrocannabinol (THC),cannabidiol (CBD), olivetol, cannabinol (CBN), cannabigerol (CBG),cannabichromene (CBC), cannabicyclol (CBCL), nabilone,tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA),cannabicyclolic acid (CBCLA), cannabigerolic acid (CBGA), cannabidiolicacid (CBDA), cannabinolic acid (CBNA), tetrahydrocannabivarin (THCV),cannabivarin (CBV), cannabidivarin (CBDV), cannabigerovarin (CBGV),cannabichromevarin (CBCV), cannabicyclovarin (CBCLV),cannabicyclovarinic acid (CBCLVA), cannabigerovarinic acid (CBGVA),tetrahydrocannabivarinic acid (THCVA), cannabichrome varinic acid(CBCVA), cannabidivarinic acid (CBDVA), as well as the prodrugs andpharmaceutically acceptable salts of these cannabinoids. Exemplaryprodrugs include alkyl ethers, haloalkyl ethers, alkyl esters, haloalkylesters, and aromatic esters, for example CBD difluoromethyl ether or CBDmethyl ether.

As used herein, the term “cannabinoid varin compound” refers tocannabinoid compounds comprising a propyl side chain attached to anaromatic ring. In one embodiment, the cannabinoid varin compound ispsychoactive. In another embodiment, the cannabinoid varin compound isnon-psychoactive. Non-limiting examples of cannabinoid varin compoundsinclude tetrahydrocannabivarin (THCV), cannabivarin (CBV),cannabidivarin (CBDV), cannabigerovarin (CBGV), cannabichromevarin(CBCV), cannabicyclovarin (CBCLV), cannabicyclovarinic acid (CBCLVA),cannabigerovarinic acid (CBGVA), tetrahydrocannabivarinic acid (THCVA),cannabichromevarinic acid (CBCVA), and cannabidivarinic acid (CBDVA), aswell as natural or synthetic molecules that have a basic cannabinoidvarin structure and are modified synthetically to provide a cannabinoidanalog. The chemical structures of exemplary cannabinoids varincompounds are shown in FIG. 2.

As used herein, the term “biphasic” refers to a system for production ofcannabinoids, which comprises two phases of solvents—a first phase and asecond phase. A solvent is the substance in which a solid, liquid, orgas is dissolved. In one embodiment, the second phase comprises anaqueous solvent, while the first phase comprises a solvent that iswater-immiscible with the aqueous solvent of the second phase. In someembodiments, the first phase forms the bottom or lower phase and thesecond phase forms the upper or top phase. In another embodiment, thesecond phase forms the bottom or lower phase and the first phase formsthe upper or top phase.

In one embodiment, the first phase comprises one or more organicsolvents that are water-immiscible or substantially water-immiscible. Inone embodiment, when the composition is agitated prior to use, themixture obtained has an opaque character. In one embodiment, the firstand second phases may be layered with one phase on top of the other. Inanother embodiment, the phases may also be arranged in an alternativeway, e.g., forming spherical or oval shapes droplets or microdropletswithin the other phase.

Each of the first phase and the second phase comprise one or moresolvents. In one embodiment, the one or more solvents comprise aco-solvent. In one embodiment, the co-solvent is an organic solvent thatcan contain one or more polar groups, such as —OH, —SH, —COOH,—C(O)R_(x), or —C(O)OR_(x), where R_(x) is a (C₁-C₅) alkyl group.Exemplary co-solvents used in the bi-phasic system include withoutlimitation, methanol, ethanol, iso-propanol, butanol, pentanol, pentane,hexane, heptane, pentene, 1,4-butane diol, dimethyl sulfoxide dimethylacetamide, dimethyl formamide, small chain fatty acid, a medium chainfatty acid, myrcene, β-caryophyllene, limonene (dipentene), α-pinene,β-pinene, citral, carvone, myrcene, citronellol, eugenol, terpinene,menthol, terpineol, terpinolene, humulene, phytol, α-phellandrene,delta-3-carene, nerol, and linalool.

As used herein, the term “microdroplet” refers to a droplet having avolume in the range from about 1 picoliter to 1 microliter. In someembodiments, droplets with a volume of 1 nanoliter to 999 nanoliters mayalso be referred to as nanodroplets. In some embodiments, themicrodroplet is formed within a biphasic system.

As used herein, the term “agitate” or “agitation” refers to mechanicalmovement, for example, rotating, vibrating, vortexing, swirling,shaking, ultrasonicating, stirring, or any movement that causes mixing.Mechanical movements include movements performed by hand or by arotator.

As used herein, the term “water-immiscible solvent” refers to anynon-aqueous or hydrophobic solvent which separates from solution intotwo distinct phases when mixed with water. The water-immiscible liquidis generally non-polar, with the non-limiting examples of thewater-immiscible liquid including terpenes, sesquiterpenes, butanone,butyl acetate, heptane, hexane, toluene, cyclohexane, petroleum ether(60-80), petroleum ether (80-100), petroleum ether (100-120), dibutylether, dipentyl ether, hexadecane, tetrachloroethylene, 1,1,1trichloroethane, mineral oil, vegetable oil, soybean oil, refinedkerosene, diesel oil, paraffin oil, white spirit or aviation crude oil,oil of an oil-based paint, grease, solvent-born or solvent-free epoxysystems, thin film and powder coating, or other water-immiscible liquidswell known in the art.

In some embodiments, the water-immiscible liquid comprises one or moreof olive oil, sesame oil, castor oil, cotton-seed oil, soybean oil,linseed oil, hemp oil, butane, pentane, heptane, octane, isooctane,nonane, decane, terpenes, di-terpenes, tri-terpenes, myrcene,β-caryophyllene, limonene, terpeneol, and the combination thereof. Insome embodiments, the water-immiscible solvent comprises acetaldehyde,acetic acid, acetone, acetonitrile, 1,2-butanediol, 1,3-butanediol,1,4-butanediol, 2-butoxyethanol, butyric acid, diethanolamine,diethylenetriamine, dimethylformamide, dimethoxyethane, dimethylsulfoxide, 1,4-dioxane, ethanol, ethylamine, ethylene glycol, formicacid, furfuryl alcohol, glycerol, methanol, methyl diethanolamine,methyl isocyanide, 1-propanol, 1,3-propanediol, 1,5-pentanediol,propanol, propanoic acid, propylene glycol, pyridine, tetrahydrofuran,and triethylene glycol.

As used herein, the term “immiscible” means a solvent or a substance(e.g., a compound, a molecule, a protein) is insoluble in a separatesolvent. The term “substantially immiscible” means that only smallamounts of the solvent or substance (e.g., a compound, a molecule, aprotein) are soluble in a separate solvent. In one embodiment, theimmiscible or substantial immiscible solvents, when mixed together,cause phase separation and form a liquid-liquid interface in between.The solubility between the two solvents can be measured by mass, weight,volume, or other unites. In one embodiment, the solubility between twosubstantially immiscible solvents at ambient temperatures (e.g., 15°C.˜25° C.) is less than 10% by weight, less than 5% by weight, or lessthan 1% by weight. In one embodiment, the solubility between twosubstantially immiscible solvents at ambient temperatures (e.g., 15°C.˜25° C.) is less than 10% by mass, less than 5% by mass, or less than1% by mass.

For instance, the phrase “substantially immiscible” refers to a firstsolvent that is partially miscible or soluble in a second solvent in arange less than 10% by weight, mass, or volume.

For example, assuming there are two solvents (solvent 1 and solvent 2),the moe fraction of solvent 1 in solvent 2 is computed as follows:

${{mole}\mspace{14mu} \% \left( {{solvent}\mspace{14mu} 1} \right)} = \frac{\eta \; \left( {{solvent}\mspace{14mu} 1} \right)}{{\eta \left( {{solvent}\mspace{14mu} 1} \right)} + {\eta \left( {{solvent}\mspace{14mu} 2} \right)}}$

The mole fraction of solvent 2 in the solvent 1 is computed as follows:

${{mole}\mspace{14mu} \% \; \left( {{solvent}\mspace{14mu} 2} \right)} = \frac{\eta \left( {{solvent}\mspace{14mu} 2} \right)}{{\eta \; \left( {{solvent}\mspace{14mu} 1} \right)} + {\eta \left( {{solvent}\mspace{14mu} 2} \right)}}$

In one embodiment, an immiscible or substantially immiscible solventrefers to a solvent that is insoluble or substantially insoluble inwater. In another embodiment, the immiscible solvent comprises anon-polar solvent.

The term “miscible” means a solvent or a substance that is soluble in aseparate solvent. In one embodiment, the separate solvent is water. Inone embodiment, the miscible solvent comprises a polar solvent.

As used herein, the term “organic solvent” refers to a hydrocarbon-basedsolvent. The organic solvent of this disclosure does not include hexane.In one embodiment, the organic solvent contains one or more polargroups. In some embodiments, the organic solvent is capable ofdissolving a substance that has low solubility in water. In oneembodiment, the organic solvents comprise one or more of olive oil,sesame oil, castor oil, cotton-seed oil, soybean oil, linseed oil, hempoil, butane, pentane, heptane, octane, isooctane, nonane, decane,terpenes, di-terpenes, tri-terpenes, myrcene, β-caryophyllene, limonene,terpeneol, and the combination thereof. In another embodiment, theorganic solvents comprises dimethyl sulfoxide (DMSO), dimethylacetamide(DMA), dimethylformamide (DMF), isopropyl alcohol, cyclodextrin, andmethanol (MeOH), dimethyl isosorbide (DMI), glycerol, propylene glycol,hexylene glycol, diethylene glycol, propylene glycol n-alkanols,1-menthol, dioxolane, ethylene glycol, other glycols, oleyl alcohol,alpha-hydroxy acids (e.g., lactic acid and glycolic acid), methyldodecyl sulfoxide, dimethylacetamide, azone(1-dodecylazacycloheptan-2-one), 2-(n-nonyl)-1,3-dioxolane, alkanols,dialkylamino acetates, or the combination thereof. In one embodiment,the organic solvent comprises terpenes, di-terpenes, tri-terpenes,myrcene, β-caryophyllene, and combinations thereof. In one embodiment,the second phase of the biphasic system comprises the organic solvent.

As used herein, the term “terpene” refers to a class of organiccompounds, derived biosynthetically from units of isoprene (C₅H₈) and totheir variants, particularly oxygenated derivatives thereof (oftencalled terpenoids). Non-limiting examples of terpenes includehemiterpenes (e.g., isoprene, prenol, and isovaleric acid), monoterpenes(e.g., myrcene, geraniol, limonene, terpineol, pinene (α- and β-pinene),menthol, thymol, carvacrol, camphor, borneol, and eucalyptol),sesquiterpenes (e.g., humulene, beta-caryophylene, neurolidol,farnesenes, and farnesol), diterpenes (e.g., cafestol, kahweol,cembrene, and taxadiene), sesterterpenes (e.g., geranylfarnesol),triterpenes, sesquarterpenes (e.g., ferrugicadiol andtetraprenylcurcumene), tetraterpenes (e.g., acyclic lycopene, themonocyclic gamma-carotene, and the bicyclic alpha- and beta-carotenes),polyterpenes, and norisoprenoids. Limonene is also called dipentene. Inone embodiment, terpenes have 10 carbon atoms or 15 carbon atoms(monoterpenes and sesquiterpenes) and oxygenated derivatives thereof. Inanother embodiment, terpene mixtures of the invention can contain smallamounts, i.e., less than 2% by weight or less than 1% by weight ofterpenes other than monoterpenes and sesquiterpenes and oxygenatedderivatives thereof. In one embodiment, the terpene is dipentene.

As used herein, the term “peroxide scavenger” refers to a component or achemical that is capable of removing or reducing peroxide or decreasingthe undesirable effects of peroxide. Non-limiting examples of peroxidescavengers include catalase, glutathione peroxidases (GPx),thioredoxin-assisted peroxidases (Prx), Sodium pyruvate, andN,N′-dimethylthiourea (DMTU). In one embodiment, the peroxide scavengercomprises catalase.

The terms “lyophilized” and “lyophilization” as used interchangeablyherein, refer to a freeze-dried process known in the art. In someembodiments, during the process a material (e.g., an enzyme) is firstfrozen and then the ice or frozen solvent is removed by sublimation in avacuum environment. An excipient may be included in pre-lyophilizedformulations to enhance stability of the lyophilized product uponstorage.

The terms “purification” or “purifying” as used interchangeably herein,refer to increasing the degree of purity of a substance of interest(e.g., an enzyme, a protein, or a compound), from a sample comprisingthe substance of interest. Methods for purification are well known inthe art. Non-limiting examples of purification methods include silicagel column chromatography, size exclusion chromatography, hydrophobicinteraction chromatography, ion exchange chromatography (e.g., cationand anion exchange chromatographies), free-flow-electrophoresis, HPLC(high performance liquid chromatography), and differentialprecipitation. In one embodiment, Sepharose SP Fast Flow resin (GEhealthcare life science) is used to purify the substance of interest(e.g., an enzyme).

The terms “recover” or “recovery” refer to a process of isolating aproduct from a reaction or a synthesis process for the product. Theproduct can be a compound, a protein, a nucleotide, or a lipid. In oneembodiment, the product recovered from the synthesis process is acannabinoid compound. The methods to recover the end products are wellknown in the art. Non-limiting examples of recovery methods includechromatography (e.g., silica gel chromatography or HPLC), activatedcharcoal treatment, filtration, distillation, precipitation, drying,chemical derivation, or combinations of these methods.

The term “analog” refers to a compound that is structurally related tonaturally occurring cannabinoids, but its chemical and/or biologicalproperties may differ from naturally occurring cannabinoids. In someembodiments, analog or analogs refer to compounds that may not exhibitone or more unwanted side effects of a naturally occurring cannabinoid.Analog also refers to a compound that is derived from a cannabinoid bychemical, biological, or a semi-synthetic transformation of thecannabinoid. The cannabinoid can be a naturally occurring, biosynthetic,or a chemically synthesized compound.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, pegylated derivatives of a parent compound, andN-oxides of a parent compound.

Unless otherwise indicated, “stereoisomer” means one stereoisomer of acompound that is substantially free of other stereoisomers of thatcompound. Thus, a single stereoisomer of a compound will besubstantially free of the other stereoisomers. A stereomerically purecompound having two chiral centers will be substantially free of otherdiastereomers of the compound. A typical stereomerically pure compoundcomprises greater than about 80% by weight of one stereoisomer of thecompound and less than about 20% by weight of other stereoisomers of thecompound, for example, greater than about 90% by weight of onestereoisomer of the compound and less than about 10% by weight of theother stereoisomers of the compound, or greater than about 95% by weightof one stereoisomer of the compound and less than about 5% by weight ofthe other stereoisomers of the compound, or greater than about 97% byweight of one stereoisomer of the compound and less than about 3% byweight of the other stereoisomers of the compound, or greater than about99% by weight of one stereoisomer of the compound and less than about 1%by weight of the other stereoisomers of the compound.

Enzymes are very specific with respect to the type of chemical reactionsthey catalyze and the nature and type of substrates that are involved inthese reactions. Enzymes also exhibit a high level of stereospecificity,regiospecificity, and chemoselectivity. It was therefore unexpected,when the present inventors observed that the purity and efficiency ofproducing cannabinoid products with the methods of this disclosure canvary, depending on the conditions under which the cannabinoid synthaseenzymes catalyze the conversion of a substrate (or precursor) to acannabinoid product.

Accordingly, the effects of temperature, pH, different solvents, ionicstrength, and/or incubation times on the distribution ratio ofcannabinoid products (e.g., the ratio of THCVA to CBCVA, THCA to CBCA,or CBDVA to THCVA and CBCVA) are provided in this disclosure. Forexample, the effect of solvent on cannabinoid product distribution ratiois evaluated.

Cannabinoids are lipophilic in nature and are poorly solubilized inaqueous solvents. The poor solubility of cannabinoids in aqueous solventhas prevented the development of ex vivo enzyme catalyzed methodologiesfor the synthesis of cannabinoids and cannabinoid analogs. The presentinvention addresses these issues by using a biphasic solvent system.Accordingly, the enzyme substrates, namely CBGA or CBGVA are dissolvedin a water-immiscible or substantially water-immiscible solvent while anappropriate cannabinoid synthase enzyme is dissolved in an aqueousbuffer. In one embodiment, the water-immiscible or substantiallywater-immiscible solvent is an organic solvent.

In one embodiment, the cannabinoid precursor is a compound of Formula I:

wherein R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b) group;R₁ and R₂ are each independently selected from the group consisting of—H, —C(O)R_(a), —OR_(a), an optionally substituted C₁-C₁₀ linear orbranched alkylene, an optionally substituted C₂-C₁₀ linear or branchedalkenylene, an optionally substituted C₂-C₁₀ linear or branchedalkynylene, an optionally substituted C₃-C₁₀ aryl, an optionallysubstituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a), and C₁-C₁₀ linear or branched alkyl; and R_(a) and R_(b) areeach independently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branchedalkyl, or a C₃-C₁₀ cycloalkyl.

In one embodiment, the cannabinoid precursor is a compound of FormulaII:

wherein R₁ is H or —COOH and R₂ is a linear or branched CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₀, C₆H₁₃, C₇H₁₅ or C₈H₁₇ group. In another embodiment, R₂ is alinear C₃H₇ or C₅H₁₀. In another embodiment, the cannabinoid precursoris CBGVA, CBGA, or their derivatives or analogs.

The present inventors surprisingly found that THCA synthase and CBDAsynthase retained their catalytic activity in a biphasic systemcomprising a first phase and a second phase. The second phase is adistinct aqueous phase. The first phase is water-immiscible with theaqueous phase of the second phase. Each of the first phase and secondphase can comprise one or more solvents. Illustrative examples of suchsolvents are dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),dimethyl acetamide (DMA), isopropyl alcohol, (IPA), methanol andcyclodextrin.

Cannabinoid compounds encompassed by the invention comprise pentyl chainand propyl chain cannabinoids. In one embodiment, the cannabinoidcompound is a pentyl chain cannabinoid. Non-limiting examples of thecannabinoid compounds include tetrahydrocannabinol (THC), cannabidiol(CBD), olivetol, cannabinol (CBN), cannabigerol (CBG), cannabichromene(CBC), cannabicyclol (CBCL), nabilone, tetrahydrocannabinolic acid(THCA), cannabichromenic acid (CBCA), cannabicyclol acid (CBCLA),cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), cannabinolic acid(CBNA), as well as the prodrugs and pharmaceutically acceptable salts ofthese cannabinoids. The prodrugs include but are not limited to alkylethers, haloalkyl ethers, alkyl esters, haloalkyl esters, andpoly-ethylene glycol ethers and esters of cannabinoids. In oneembodiment, the prodrug of CBD is a CBD difluoromethyl ether or a CBDmethyl ether compound. In certain embodiments, the cannabinoid compoundis nabilone, dronabinol, anandamide as well as natural or syntheticmolecules that have a basic cannabinoid structure and are modifiedsynthetically to provide a cannabinoid analog.

In another embodiment, the cannabinoid compound is a cannabinoid havinga propyl side chain attached to an aromatic ring, also known as a“varin”. While varins are present in the cannabis plant, their naturalabundance in plant tissue is low. For example, the natural abundance ofseveral varin compounds in plant tissue is between 0.5%-1.5%. By using abiphasic solvent system, the present invention permits synthesis ofseveral varin compounds in high volumetric yields. Non-limiting examplesof varin compounds synthesized using the biphasic solvent system andmethods of the invention include tetrahydrocannabivarin (THCV),cannabivarin (CBV), cannabidivarin (CBDV), cannabigerovarin (CBGV),cannabichrome varin (CBCV), cannabicyclovarin (CBCLV),cannabicyclovarinic acid (CBCLVA), cannabigerovarinic acid (CBGVA),tetrahydrocannabivarinic acid (THCVA), cannabichromevarinic acid(CBCVA), cannabidivarinic acid (CBDVA), as well as natural or syntheticmolecules that have a basic cannabinoid varin structure and are modifiedsynthetically to provide a cannabinoid analog. FIG. 2 shows the chemicalstructures of some cannabinoid compounds.

Methods of Producing Cannabinoid Products

One aspect of the invention provides a method of producing a cannabinoidproduct. In some embodiment, the cannabinoid product comprises pentylchain or propyl chain cannabinoids. In one embodiment, the cannabinoidproduct comprises a cannabinoid varin.

In one embodiment, the method for producing a cannabinoid productcomprises contacting a cannabinoid precursor in a first phase with acannabinoid synthase in a second phase. The cannabinoid precursor is asubstrate of a cannabinoid synthase.

In one embodiment, the cannabinoid precursor is a compound of Formula I:

wherein R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b) group;R₁ and R₂ are each independently selected from the group consisting of—H, —C(O)R_(a), —OR_(a), an optionally substituted C₁-C₁₀ linear orbranched alkylene, an optionally substituted C₂-C₁₀ linear or branchedalkenylene, an optionally substituted C₂-C₁₀ linear or branchedalkynylene, an optionally substituted C₃-C₁₀ aryl, an optionallysubstituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a), and C₁-C₁₀ linear or branched alkyl; and R_(a) and R_(b) areeach independently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branchedalkyl, or a C₃-C₁₀ cycloalkyl.

In one embodiment, the cannabinoid precursor is a compound of formulaII:

wherein R₁ is H or —COOH and R₂ is a linear or branched CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₀, C₆H₁₃, C₇H₁₅, or C₈H₁₇ group. In another embodiment, R₂ isa linear C₃H₇ or C₅H₁₀. In another embodiment, the cannabinoid precursoris CBGVA, CBGA or derivatives or analogs of CBGA and CBGVA.

In one embodiment, the first phase comprise an organic solvent and thesecond phase comprises an aqueous solvent. In one embodiment, the firstphase and the second phase are substantially immiscible or immiscible,and thus the method of this disclosure comprises a biphasic process.

In some embodiments, the methods further comprise agitating the organicsolvent to form micro-droplets within the aqueous solution, wherein atleast one micro-droplet comprises the cannabinoid precursor. In oneembodiment, the cannabinoid precursor is CBGVA, CBGA, and theirderivative or analog.

The size of the microdroplet can vary depending on the solvents, themethod or orientation of agitation, or the composition within eachsolvent. In some embodiments, the microdroplet has a volume ranging lessthan 1 picoliter, between 1 picoliter to 1 microliter, or above 1microliter. The duration of agitation can vary as well. In oneembodiment, the duration of agitation is less than 5 seconds, 10seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 10 hours, or24 hours. In some embodiments, the duration of agitation is more than 24hours.

In one embodiment, the first phase comprises an organic solvent, whichcan be polar or non-polar. In another embodiment, the organic solvent issubstantially water-immiscible. In some embodiments, the first phase iscapable of dissolving a substance that has low solubility in water. Inone embodiment, the first phase comprises one or more of olive oil,sesame oil, castor oil, cotton-seed oil, soybean oil, linseed oil, hempoil, butane, pentane, heptane, octane, isooctane, nonane, decane,terpenes, di-terpenes, tri-terpenes, myrcene, β-caryophyllene, limonene,and terpeneol. In another embodiment, the terpene comprises one of moreof hemiterpene, monoterpene, sesquiterpene, diterpene, sesterterpene,triterpene, sesquarterpene, tetraterpene, polyterpene, andnorisoprenoid. In another embodiment, the terpene comprises one or moreof di-terpenes, tri-terpenes, myrcene, β-caryophyllene, limonene,pinene, and linalool. In one embodiment, the first phase may comprisefatty acids or fatty acid esters.

In another embodiment, the first phase comprises one or more of mineraloil, vegetable oil, refined kerosene, diesel oil, paraffin oil, or otherwater-immiscible liquids well known in the art. In one embodiment, thefirst phase further comprises, acetic acid, acetone, acetonitrile,1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, butyricacid, diethanolamine, diethylenetriamine, dimethylformamide,dimethoxyethane, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethylamine,ethylene glycol, formic acid, furfuryl alcohol, glycerol, methanol,methyl diethanolamine, methyl isocyanide, 1-propanol, 1,3-propanediol,1,5-pentanediol, propanol, propanoic acid, propylene glycol, pyridine,tetrahydrofuran, and triethylene glycol. In another embodiment, thefirst phase comprises terpene. In another embodiment, terpene comprisesone of more of hemiterpene, monoterpene, sesquiterpene, diterpene,sesterterpene, triterpene, sesquarterpene, tetraterpene, polyterpene,and norisoprenoid. In another embodiment, the terpene comprises one ormore of di-terpenes, tri-terpenes, myrcene, β-caryophyllene, limonene(dipentene), α-pinene, β-pinene, citral, carvone, myrcene, citronellol,eugenol, terpinene, menthol, terpineol, terpinolene, humulene, phytol,α-phellandrene, delta-3-carene, nerol, and linalool. In one embodiment,terpenes have 10 carbon atoms or 15 carbon atoms (monoterpenes andsesquiterpenes) and oxygenated derivatives thereof. In anotherembodiment, terpene mixtures of the invention can contain small amounts,i.e., less than 2% by weight or less than 1% by weight of terpenes otherthan monoterpenes and sesquiterpenes and oxygenated derivatives thereof.In another embodiment, the first phase comprises fatty acids, fatty acidesters, or the combination thereof.

The first phase may contain a co-solvent. The amount of co-solvent inthe first phase depends on the composition, the concentration, pH,temperature, or other conditions. In one embodiment, the amount ofco-solvent within the first phase is less than 50%, in the range betweenabout 5% and about 49%, between about 10% and about 49%, between about20% and about 49%, between about 30% and about 49%, between about 40%and about 49%, or between about 45% and about 49%. In one embodiment,the amount of co-solvent is between about 30% and about 49%. In anotherembodiment, the amount of co-solvent is between about 10% and about 25%,or 2% to 15%.

In another embodiment, the solvent of the first phase comprises soybeanoil. In some embodiments, the amount of soybean oil is greater than 50%,in the range between about 51% and about 90%, between about 60% andabout 80%, between about 70% and about 79%, between about 80% and about90%, between about 90% and about 100%, or between about 50% and about99% of the organic solvent. In another embodiment, the amount of soybeanoil is between about 50% and about 60%. In some embodiments, the amountof soybean oil is about 90%.

In another embodiment, the organic solvent of the first phase comprisesterpene. In some embodiments, the amount of terpene is greater than 50%,in the range between about 50% and about 90%, between about 60% andabout 80%, between about 70% and about 79%, between about 80% and about90%, between about 90% and about 100%, or between about 50 and about 55%of the organic solvent.

In another embodiment, the organic solvent of the first phase compriseslimonene (or dipentene). In some embodiments, the amount of limonene isgreater than 50%, in the range between about 50% and about 90%, betweenabout 60% and about 80%, between about 70% and about 79%, between about80% and about 90%, between about 50% and about 60%, or between about 55%and 65% of the organic solvent.

The enzymatic efficiency of cannabinoid synthase (e.g., THCA synthase orCBDA synthase) can be affected by the types and concentrations ofco-solvent in the second phase that comprises an aqueous solvent. Thus,in one embodiment, the second phase comprises an aqueous miscibleco-solvent. The aqueous miscible co-solvent, in some embodiments,comprises one or more of dimethyl sulfoxide (DMSO), dimethylacetamide(DMA), dimethylformamide (DMF), ethanol, isopropyl alcohol,cyclodextrin, peroxide scavenger, and methanol (MeOH), and thecombination thereof. In some embodiments, the amount of the aqueousmiscible co-solvent in the second phase is less than 0.1%, between about0.1% and about 49% (w/v), about 1% and about 49%, about 5% and about49%, or about 10% and about 49%, or about 20% and about 49%, about 30%and about 49% of the aqueous solution. In some embodiments, the amountof the aqueous miscilble co-solvent in the second phase is above 0.1%,1%, 10%, 20%, 30%, 40%, or 49%

In some embodiments, the second phase comprises dimethyl sulfoxide(DMSO) as a water miscible co-solvent in an amount less than 0.1%,between about 0.1% and about 49% (w/v), about 1% and about 49%, about 5%and about 49%, or about 10% and about 49%, or about 20% and about 49%,or about 30% and about 49% of the aqueous solution. In one embodiment,the amount of DMSO in the second phase is above 0.1%, 1%, 10%, 20%, 30%,40%, or 49%. In another embodiment, the second phase comprises DMSO inan amount between about 1% and about 20% of the aqueous solution. In oneembodiment, the amount of DMSO is between about 10% and about 20%. Inanother embodiment, the amount of DMSO is about 20%.

In one embodiment, the second phase comprises water miscible co-solventmethanol (MeOH) in an amount less than 0.1%, between about 0.1% andabout 49% (w/v), about 1% and about 49%, about 5% and about 49%, orabout 10% and about 49%, or about 20% and about 49%, or about 30% andabout 49% of the aqueous solution. In one embodiment, the amount of MeOHin the second phase is above 0.1%, 1%, 10%, 20%, 30%, 40%, or 49%. Inanother embodiment, the second phase comprises MeOH in an amount betweenabout 1% and about 20% of the aqueous solution. In a differentembodiment, the amount of MeOH is between about 10% and about 20%. In adifferent embodiment, the amount of MeOH is between about 1% and about6%.

In one embodiment, the second phase comprises water miscible co-solventdimethylacetamide (DMA) in an amount less than 0.1%, between about 0.1%and about 49% (w/v), about 1% and about 49%, about 5% and about 49%, orabout 10% and about 49%, or about 20% and about 49%, or about 30% andabout 49% of the aqueous solvent. In one embodiment, the amount of DMAis above 0.1%, 1%, 10%, 20%, 30%, 40%, or 49%. In another embodiment,the second phase comprises DMA in an amount between about 1% and about20% of the aqueous solvent. In a different embodiment, the amount of DMAis between about 10% and about 20%. In a different embodiment, theamount of DMA is between about 1% and about 6%.

Surprisingly, Applicant discovered that inclusion of a peroxidescavenger increased the enzymatic activity of cannabinoid synthase. Inone embodiment, the peroxide scavenger comprises one or more ofcatalase, glutathione peroxidases (GPx), thioredoxin-assistedperoxidases (Prx), Sodium pyruvate, and N,N′-dimethylthiourea (DMTU). Inanother embodiment, the peroxide scavenger is catalase. In oneembodiment, the amount of the peroxide scavenger in the aqueous solutionis between about 0.001% and about 0.1%, about 0.005% and about 0.05%, orabout 0.01% and about 0.03% (w/v). In one embodiment, the amount ofperoxide scavenger is about 0.01%. In another embodiment, the amount ofcatalase in the aqueous solvent is between about 0.001% and about 0.1%,about 0.005% and about 0.05%, or about 0.01% and about 0.03% (w/v). Inanother embodiment, the amount of catalase is about 0.01% of the aqueoussolution.

The inventors were surprised to observe that the ratio of cannabinoidand the low abundance of varin compounds is altered by the pH of theaqueous phase that contains the cannabinoid synthase. In one embodiment,the pH of the aqueous solution ranges from about 3.5 to about 10.0, fromabout 3.5 to about 9, from about 4 to about 8, or from about 5.5 toabout 7.5. In one embodiment, the pH value ranges from about 3.5 toabout 9.0. In one embodiment, the pH value ranges from about 4.5 toabout 7.5. Alternatively, the pH value ranges from about 5.5 to about7.5. In some embodiments, the pH value ranges from about 5.0 to about6.5. In one embodiment, the pH value is about 7.5. In anotherembodiment, the pH value is about 5.5. In yet another embodiment, the pHvalue is about 4.5.

The amount of a water miscible organic co-solvent can affect theproduction of cannabinoid compounds. In some embodiments, the aqueoussolution comprises DMSO in a range between about 1% and about 30%, about2% and about 20%, or about 5% and about 10% of the aqueous solution,wherein the pH value of the aqueous solution is between about 3 andabout 9, about 4 and about 8, or about 5.5 and about 7.5. In someembodiments, the aqueous solution comprises DMSO in a range betweenabout 5% and about 10% of the aqueous solution, wherein the pH value ofthe aqueous solution is between about 5.5 and about 7.5. In oneembodiment, the aqueous solution comprises DMSO in an amount of about5%, wherein the pH value of the aqueous solution is about 5.5. Inanother embodiment, the aqueous solvent comprises DMSO in an amount ofabout 10%, wherein the pH value of the aqueous solution is about 7.5.

In one embodiment, the volume ratio of the first phase to the secondphase is from about 1:9 to about 9:1; from about 1:8 to about 8:1; fromabout 1:7 to about 7:1; from about 1:6 to about 6:1; from about 1:5 toabout 5:1; from about 1:4 to about 4:1; from about 1:3 to about 3:1; orfrom about 1:2 to about 2:1. In one embodiment, the volume ratio is fromabout 1:9 to about 9:1. In another embodiment, the volume ratio is fromabout 1:2 to about 2:1.

The methods of this disclosure use a cannabinoid synthase as catalystfor synthesizing the cannabinoid compound. In one embodiment, thecannabinoid synthase comprises cannabidiolic acid synthase (CBDAsynthase), a tetrahydrocannabinolic acid synthase (THCA synthase), or acannabichromene acid synthase (CBCA synthase). In one embodiment, thecannabinoid synthase comprises CBDA synthase or THCA synthase. In oneaspect of the invention, the cannabinoid synthase is dissolved in anaqueous phase. The cannabinoid synthase can be purified from plant(e.g., C. sativa). The synthase can also be produced in eithereukaryotic or prokaryotic cells, e.g., E coli, yeast, baculovirus hosts,mammalian cells, algae, tobacco plant cells in culture, or insect cells.The methods for expressing recombinant cannabinoid synthases aredisclosed in WO2014134281, which is incorporated by reference in itsentirety. In one embodiment, the cannabinoid synthase used for themethod can be in its crude form or its purified form before dissolvingin a solvent.

In another embodiment, the cannabinoid synthase (e.g., CBDA synthase orTHCA synthase) is secreted into the culture medium in which theeukaryotic or prokaryotic cells are grown. For example, the codingsequence of the gene coding for the cannabinoid biosynthetic enzyme isoperably linked to a secretion signal. For a yeast (e.g., Pichia), thesignal sequence could be an alpha factor secretion signal. After thecannabinoid biosynthetic enzyme is secreted into the yeast growthmedium, the yeast cells are removed and the growth medium is lyophilized(freeze dried) following filtration of the growth medium containing thecannabinoid synthase enzyme using a 10K molecular weight filter. Themethods for expressing recombinant THCA synthase and CBDA synthase aredescribed in WO2014134281, which is incorporated by reference in itsentirety. Recovering enzyme in the lyophilized medium resulted in about4% of the lyophilized medium comprising THCA synthase or CBDA synthase.Accordingly, in the working examples contained herein, if 100 grams oflyophilized medium was used as the technical grade enzyme, the mediumcontained about 4 grams of either THCA synthase or CBDA synthase.

The concentration of the synthases can vary based on the concentrationsof substrates, the reaction conditions, or the target products. In oneembodiment, the cannabinoid synthase used is a purified synthase.Without being bound by a theory, the requisite concentrations ofpurified synthase are normally less than those of crude synthase. In oneembodiment, the concentration of the synthase in the aqueous solution isat least 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 5 mg/mL, 10 mg/mL, 32mg/mL, 50 mg/mL, or 100 mg/mL. In another embodiment, the concentrationof the synthase in the aqueous solution is at least 5 mg/mL. In anotherembodiment, the concentration of the synthase in the aqueous solution isat least 32 mg/mL. In one embodiment, the concentration of the purifiedsynthase in the aqueous solution is at least 50 μg/mL. In someembodiments, the concentration of the purified synthase in the aqueoussolution is at least 200 μg/mL.

In some embodiments, the concentration of cannabinoid precursor in theorganic solvent is at least about 0.1 mg/mL, 1 mg/mL, 10 mg/mL, about 50mg/mL, about 100 mg/mL, about 150 mg/mL, about 200 mg/mL, about 250mg/mL, or about 300 mg/mL. In one aspect of this invention, theconcentration of cannabinoid precursor in the organic solvent is betweenabout 0.1 mg/mL and about 250 mg/mL, about 1 mg/mL and 200 mg/mL, about20 mg/mL and about 150 mg/mL, or about 50 mg/mL and about 100 mg/mL. Insome embodiment, the cannabinoid precursor is CBGA, CBGVA, or theirderivatives or analogs.

The progress of the reaction can be monitored periodically orcontinuously. For example, the decrease in the concentration ofcannabinoid precursor (e.g., CBGVA and CBGA) can be monitored to signaltermination of synthesis. Alternatively, reaction progress is monitoredby monitoring the formation of a cannabinoid, for examplespectrophotometrically. Once the synthesis is terminated, thecannabinoid product thus produced can be readily recovered from themedium using standard solvent extraction or chromatographic purificationmethods. Thus, the methods of this disclosure further compriserecovering the cannabinoid composition or product.

In one embodiment, the recovered cannabinoid product comprises acannabinoid varin compound (propyl side chain cannabinoid), whichinclude one or more of tetrahydrocannabivarin (THCV), cannabivarin(CBV), cannabidivarin (CBDV), cannabigerovarin (CBGV), cannabichromevarin (CBCV), cannabicyclovarin (CBCLV), cannabicyclovarinic acid(CBCLVA), cannabigerovarinic acid (CBGVA), tetrahydrocannabivarinic acid(THCVA), cannabichromevarinic acid (CBCVA), cannabidivarinic acid(CBDVA), cannabichrome varinic acid (CBCVA), cannabidivarinic acid(CBDVA), or its analogs or derivatives. In one embodiment, the recoveredcannabinoid product comprises THCVA, CBCVA, or both. In anotherembodiment, the recovered cannabinoid product comprises CBDVA, CBCVA,and optionally THCVA.

In one embodiment, the recovered cannabinoid product is a pentyl chaincannabinoid, which includes one or more of THC, CBD, CBN, CBG, CBC,CBCL, nabilone, THCA, CBCA, CBCLA, CBGA, CBDA, CBNA, and theirderivatives, analogs, prodrugs, or any natural or synthetic moleculesthat have a basic cannabinoid structure and are modified synthetically.In one embodiment, the recovered cannabinoid product comprises THCA,CBCA, or both. In another embodiment, the recovered cannabinoid productcomprises CBDA, CBCA, or optionally THCA. Neutral forms of thecannabinoids (e.g., THC, CBD, CBC, THCV, CBDV, CBG, and CBGV) are theresult of non-enzymatic decarboxylation by exposure to, for example:heat, light, and pH.

The methods of recovering the cannabinoid products from the disclosedreaction are disclosed in WO2014134281, which is incorporated byreference in its entirety. Non-limiting examples of recovery methodsinclude chromatography (e.g., HPLC or silica gel), activated charcoaltreatment, filtration, distillation, precipitation, drying, chemicalderivation, or combinations of these methods.

Composition

Another aspect of the disclosure relates to a composition that can beused for synthesizing the cannabinoid compounds, their analogs orderivatives. In one aspect of the invention, the composition comprises(a) a cannabinoid precursor in a first phase; and (b) a cannabinoidsynthase in a second phase.

In one embodiment, the cannabinoid precursor is a compound of Formula I:

wherein R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b) group;R₁ and R₂ are each independently selected from the group consisting of—H, —C(O)R_(a), —OR_(a), an optionally substituted C₁-C₁₀ linear orbranched alkylene, an optionally substituted C₂-C₁₀ linear or branchedalkenylene, an optionally substituted C₂-C₁₀ linear or branchedalkynylene, an optionally substituted C₃-C₁₀ aryl, an optionallysubstituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene, orR₁ and R₂ together with the carbon atoms to which they are bonded form aC₅-C₁₀ cyclic ring; R₃ is selected from the group consisting of H,—C(O)R_(a) and C₁-C₁₀ linear or branched alkyl; and R_(a) and R_(b) areeach independently —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branchedalkyl, or a C₃-C₁₀ cycloalkyl.

In one embodiment, the cannabinoid precursor is a compound of FormulaII:

wherein R₁ is H or —COOH and R₂ is a linear or branched CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₀, C₆H₁₃, C₇H₁₅ or C₈H₁₇ group. In another embodiment, R₂ is alinear C₃H₇ or C₅H₁₀. In another embodiment, the cannabinoid precursoris CBGA, CBGVA, or their derivatives or analogs. In some embodiment, thecannabinoid precursor is CBGA. In another embodiment, the cannabinoidprecursor is CBGVA.

In one embodiment, the first phase comprises an organic solvent and thesecond phase comprises an aqueous solvent. In one embodiment, theorganic solvent is water-immiscible or substantially water-immiscible.

In some embodiments, the organic solvent is capable of dissolving asubstance that has low solubility in water. The organic solvent may bepolar or non-polar. In one embodiment, the first phase comprises one ormore of olive oil, sesame oil, castor oil, cotton-seed oil, soybean oil,linseed oil, hemp oil, butane, pentane, heptane, octane, isooctane,nonane, decane, terpenes, di-terpenes, tri-terpenes, myrcene,β-caryophyllene, limonene, and terpeneol. In another embodiment, terpenecomprises one of more of hemiterpene, monoterpene, sesquiterpene,diterpene, sesterterpene, triterpene, sesquarterpene, tetraterpene,polyterpene, and norisoprenoid. In another embodiment, the terpenecomprises one or more of di-terpenes, tri-terpenes, myrcene,β-caryophyllene, limonene, pinene, and linalool. The first phasecomprises fatty acids or fatty acid esters.

In another embodiment, the first phase comprises one or more of mineraloil, vegetable oil, refined kerosene, diesel oil, paraffin oil, or otherwater-immiscible liquids well known in the art. In one embodiment, thefirst phase comprises an organic solvent selected from the groupconsisting of acetic acid, acetone, acetonitrile, 1,2-butanediol,1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, butyric acid,diethanolamine, diethylenetriamine, dimethylformamide, dimethoxyethane,dimethyl sulfoxide, 1,4-dioxane, ethanol, ethylamine, ethylene glycol,formic acid, furfuryl alcohol, glycerol, methanol, methyldiethanolamine, methyl isocyanide, 1-propanol, 1,3-propanediol,1,5-pentanediol, propanol, propanoic acid, propylene glycol, pyridine,tetrahydrofuran, and triethylene glycol.

A co-solvent may be present in the first phase that comprises an organicsolvent. The amount of co-solvent in the first phase depends on thecomposition, the concentrations, pH, temperature, or other conditions.In one embodiment, the amount of organic co-solvent within the firstphase is less than 5%, in the range between about 5% and about 49%,between about 10% and about 49%, between about 20% and about 49%,between about 30% and about 49%, between about 40% and about 49%, orbetween about 45% and about 49%. In one embodiment, the amount oforganic co-solvent is between about 30% and about 49%. In anotherembodiment, the amount of organic co-solvent is between about 45% andabout 49%. In another embodiment, the amount of organic co-solvent is atleast about 50%. In some embodiments, the amount of organic co-solventis at least about 25%.

In a preferred embodiment, the organic solvent comprising the firstphase is soybean oil. In some embodiments, the amount of soybean oil isgreater than 50%, in the range between about 50% and about 90%, betweenabout 50% and about 80%, between about 50% and about 79%, between about50% and about 70%, between about 50% and about 60%, or between about 50%and about 55% of the organic solvent. In another embodiment, the amountof soybean oil is between about 50% and about 60%. In some embodiments,the amount of soybean oil is about 53%.

In another embodiment, the organic solvent comprising the first phase isterpene. In some embodiments, the amount of terpene is greater than 50%,in the range between about 50% and about 90%, between about 50% andabout 80%, between about 50% and about 79%, between about 50% and about70%, between about 50% and about 60%, or between about 51% and about 55%of the organic solvent.

In one embodiment, the second phase comprises one or more polarco-solvents that are miscible in water. In one embodiment, the watermiscible co-solvent comprises one or more of dimethyl sulfoxide (DMSO),dimethylacetamide (DMA), dimethylformamide (DMF), isopropyl alcohol,cyclodextrin, peroxide scavenger, and methanol (MeOH), and thecombination thereof. In some embodiments, the amount of the watermiscible co-solvent in the second phase is less than 0.1%, between about0.1% and less than 50% (w/v), about 1% and about 50%, about 5% and about40%, or about 5% and about 30%, or about 5% and about 20%, about 10% andabout 15% of the aqueous solution.

In some embodiments, the second phase comprises DMSO in an amount lessthan 0.1%, between about 0.1% and about 50% (w/v), about 1% and about50%, about 5% and about 40%, or about 5% and about 30%, or about 5% andabout 20%, or about 10% and about 15% of the aqueous solution. In oneembodiment, the amount of DMSO in the second phase is above 0.1%, 1%,10%, 20%, 30%, 40%, or less than 50%. In another embodiment, the secondphase comprises DMSO in an amount between 1% and 20% of the aqueoussolution. In one embodiment, the amount of DMSO is between about 10% andabout 20%. In another embodiment, the amount of DMSO is about 20%.

In one embodiment, the second phase comprises MeOH in an amount lessthan 0.1%, between about 0.1% and about 50% (w/v), about 1% and about50%, about 5% and about 40%, or about 5% and about 30%, or about 5% andabout 20%, or about 10% and about 15% of the aqueous solution. In oneembodiment, the amount of MeOH in the second phase is above 0.1%, 1%,10%, 20%, 30%, 40%, or 49%. In another embodiment, the second phasecomprises MeOH in an amount between about 1% and about 20% of theaqueous solution. In a different embodiment, the amount of MeOH isbetween about 10% and about 20%. In a different embodiment, the amountof MeOH is between about 1% and about 6%.

In one embodiment, the second phase comprises dimethylacetamide (DMA) inan amount less than 0.1%, between about 0.1% and about 50% (w/v), about1% and about 50%, about 5% and about 40%, or about 5% and about 30%, orabout 5% and about 20%, or about 10% and about 15% of the aqueoussolvent. In one embodiment, the amount of DMA is about 0.1%, 1%, 10%,20%, 30%, 40%, 49%. In another embodiment, the second phase comprisesDMA in an amount between about 1% and about 20% of the aqueous solvent.In a different embodiment, the amount of DMA is between about 10% andabout 20%. In a different embodiment, the amount of DMA is between about1% and about 6%.

In one embodiment, the biphasic system of this invention comprises aperoxide scavenger that comprises one or more of catalase, glutathioneperoxidases (GPx), thioredoxin-assisted peroxidases (Prx), sodiumpyruvate, and N′,N′-dimethylthiourea (DMTU). In one embodiment, theperoxide scavenger is catalase. In one embodiment, the amount of theperoxide scavenger in the aqueous solution is between about 0.001% andabout 0.1%, about 0.005% and about 0.05%, or about 0.01% and about 0.03%(w/v). In one embodiment, the amount of peroxide scavenger is about0.01%. In another embodiment, the amount of catalase in the aqueoussolvent is between about 0.001% and about 0.1%, about 0.005% and about0.05%, or about 0.01% and about 0.03% (w/v). In another embodiment, theamount of catalase is about 0.01% of the aqueous solution.

As noted above, the disclosure provides that the pH value and the ratioof organic solvent to the aqueous solution in the composition canunexpectedly affect the cannabinoid synthesis and the ratio ofcannabinoid products produced. In one embodiment, the pH value of theaqueous solution in the composition ranges from about 3.5 to about 10.0,from about 3.5 to about 9, from about 4 to about 8, or from about 5.5 toabout 7.5. In one embodiment, the pH value ranges from about 4.5 toabout 7.5. Alternatively, the pH value ranges from about 5.5 to about7.5. In some embodiments, the pH value ranges from about 5.0 to about6.5. In one embodiment, the pH value is about 7.5. In anotherembodiment, the pH value is about 5.5. In yet another embodiment, the pHvalue is about 4.5.

In some embodiments, the second phase comprises DMSO in a range betweenabout 1% and 30%, about 2% and about 20%, or 5% and about 10% of theaqueous solution, wherein the pH value of the aqueous solution isbetween about 3 and about 9, about 4 and about 8, or about 5.5 and about7.5. In some embodiments, phase 2 comprises DMSO in a range betweenabout 5% and about 10% of the aqueous solution, wherein the pH value ofthe aqueous solution is between about 5.5 and about 7.5. In oneembodiment, the the second phase comprises DMSO in an amount of about5%, wherein the pH value of the aqueous solution is about 5.5. Inanother embodiment, the the second phase comprises DMSO in an amount ofabout 10%, wherein the pH value of the aqueous solution is about 7.5.

In one embodiment, the volumetric ratio of the organic solvent to theaqueous solution is from about 1:9 to about 9:1; from about 1:8 to about8:1; from about 1:7 to about 7:1; from about 1:6 to about 6:1; fromabout 1:5 to about 5:1; from about 1:4 to about 4:1; from about 1:3 toabout 3:1; or from about 1:2 to about 2:1. In another embodiment, thevolume ratio is from about 1:2 to about 2:1.

In some embodiments, the composition further comprises a cannabinoidsynthase which comprises one or more of CBDA synthase, THCA synthase,or/and CBCA synthase. In one embodiment, the composition comprises CBDAsynthase or THCA synthase. The cannabinoid synthase can be in its crudeform or its purified form. In one embodiment, the cannabinoid synthaseis lyophilized to a powder which is directly added to the second phaseor alternatively, the lyophilized powder is dissolved in a specificvolume of a buffer and this solution is added to the second phase of thebi-phasic system.

In one embodiment, the concentration of the synthase in the aqueoussolution is at least 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 5 mg/mL, 10mg/mL, 32 mg/mL, 50 mg/mL, or 100 mg/mL. In another embodiment, theconcentration of the synthase in the aqueous solution is at least 5mg/mL. In another embodiment, the concentration of the synthase in theaqueous solution is at least 32 mg/mL. For some embodiments, thecannabinoid synthase is purified and the concentration of the purifiedsynthase in the aqueous solution is at least 50 μg/mL. In someembodiments, the concentration of the purified synthase in the aqueoussolution is at least 200 μg/mL.

In some embodiments, the concentration of cannabinoid precursor in theorganic solvent is at least about 0.1 mg/mL, 1 mg/mL, 10 mg/mL, 20mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, about 50 mg/mL, about 100 mg/mL,about 150 mg/mL, about 200 mg/mL, about 250 mg/mL, or about 300 mg/mL.In one aspect of this invention, the concentration of cannabinoidprecursor in the organic solvent is between about 0.1 mg/mL and about250 mg/mL, about 1 mg/mL and 200 mg/mL, about 20 mg/mL and about 150mg/mL, or about 50 mg/mL and about 100 mg/mL. In one embodiment, thecannabinoid precursor is one or more of CBGA, CBGVA, or their derivativeor analog.

Apparatus and System

This disclosure also provides an apparatus or a system for producing oneor more cannabinoids, cannabinoid prodrugs, or cannabinoid analogs. Theapparatus may comprise a fermentor 10, a bioreactor 30, and a controlmechanism 40. FIG. 1A depicts an apparatus 100 configured to produce atleast one cannabinoid, cannabinoid prodrug and/or at least onecannabinoid analog according to an embodiment. As shown in FIG. 1A, theapparatus 100 includes a fermentor 10, a bioreactor 30, and a controlmechanism (controller) 40. The fermentor 10 holds cell culture medium 12and a plurality of cells 14. The cells 14 are configured to produce oneor more cannabinoid acid synthases. The cells may be geneticallyengineered according to the invention to secrete the cannabinoid acidsynthase into the medium. Optionally, the majority of the cannabinoidacid synthase remains intracellular, is secreted into the medium, or isfound both inside and outside the cells. The cells 14 grown in thefermentor 10 for the manufacture of a cannabinoid acid synthase can beprokaryotes such as Escherichia coli, Bacillus, Pseudomonas or anynumber of gram positive or gram negative bacteria. Alternatively, thecells 14 grown in the fermentor 10 can be eukaryotic cells such as yeast(e.g., Pichia, Saccharomyces, Yarrowia), algae, insect, or plant cells.In one embodiment, the prokaryotic or eukaryotic cells are geneticallymodified to include a nucleic acid construct comprising one or moregenes that encode a cannabinoid acid synthase protein. In oneembodiment, the cannabinoid synthase comprises CBDA synthase or THCAsynthase.

In certain embodiments, the nucleic acid sequence that encodes acannabinoid acid synthase protein is modified to include a secretionsignal operably linked to the 5′ region of the cannabinoid synthasegene. In another embodiment, cannabinoid acid synthase proteins includea 6-residue histidine tag at their 3′ end to facilitate enzymepurification. The addition of a secretion sequence permits secretion ofthe cannabinoid acid synthase protein into the medium 12 used forprokaryotic or eukaryotic cell growth. Following production of one ormore cannabinoid acid synthases in the fermentor 10, the supernatant iscollected and dried to produce a technical grade enzyme. Drying can bedone by any method known in the art such as lyophilization, freezedrying, or the like. Alternatively, the enzyme is purified using amethod well known in the art, such as nickel column chromatography, andthen introduced into the aqueous second phase.

The bioreactor 30 is designed to permit mixing of the first phase andsecond phase after introduction of substrate into the first phase andcannabinoid synthase enzyme into the second phase. The first phasecomprises a cannabinoid precursor. In some embodiments, the cannabinoidprecursor is the compound of Formula II:

wherein R₁ is H or —COOH and R₂ is a linear or branched CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₀, C₆H₁₃, C₇H₁₅ or C₈H₁₇ group, wherein the cannabinoidprecursor is configured to interact with the cannabinoid synthase toform the cannabinoids or its analog. In one embodiment, the cannabinoidprecursor is cannabigerolic acid (CBGA), cannabigerovarinic acid(CBGVA), or their derivative or analog. In another embodiment, the firstphase of the bioreactor is agitated to form micro-droplets within thesecond phase, wherein at least one micro-droplet comprises thecannabinoid precursor.

Mixing of the first and second phases is accomplished in any way knownin the art such as shaking, spinning, sparging with a gas such asoxygen, or stirring with an impeller. In one embodiment, the first phasecomprises an organic solvent and the second phase comprises an aqueoussolvent. In another embodiment, the first phase is substantiallywater-immiscible or water-immiscible. In one embodiment, thesubstantially water immiscible or water immiscible solvent comprises oneor more of olive oil, sesame oil, castor oil, cotton-seed oil, soybeanoil, butane, pentane, heptane, octane, isooctane, nonane, decane, andterpene. In another embodiment, the terpene comprises one or more ofhemiterpene, monoterpene, sesquiterpene, diterpene, sesterterpene,triterpene, sesquarterpene, tetraterpene, polyterpene, andnorisoprenoid. In another embodiment, the terpene comprises one or moreof diterpene, tri-terpene, myrcene, β-caryophyllene, limonene (ordipentene), pinene, and linalool. In one embodiment, the organic solventcomprises soybean oil. In another embodiment, the aqueous solventfurther comprises one or more of dimethyl sulfoxide (DMSO),dimethylformamide (DMF), dimethylacetamide (DMA), isopropyl alcohol,cyclodextrin, peroxide scavenger, and methanol (MeOH), wherein theamount of the aqueous solvent is between about 0.001% and about 50%(w/v), about 1% and about 40%, about 1% and about 30%, or about 1% andabout 20% of the second phase. In another embodiment, the aqueoussolvent comprises DMSO in an amount between about 0.1% and about 50% ofthe aqueous solution. In another embodiment, the aqueous solventcomprises MeOH in an amount between about 1% and about 20% of theaqueous solution. In another embodiment, the peroxide scavenger is oneor more of catalase, glutathione peroxidases (GPx), thioredoxin-assistedperoxidases (Prx), Sodium pyruvate, and N,N′-dimethylthiourea (DMTU). Inanother embodiment, the aqueous solvent comprises the peroxide scavengerin an amount between about 0.001% and about 0.1%, about 0.005% and about0.05%, or about 0.01% and about 0.03% of the aqueous solution. Inanother embodiment, the aqueous solvent comprises catalase in an amountbetween about 0.001% and about 0.1%, about 0.005% and about 0.05%, orabout 0.01% and about 0.03% of the aqueous solution. In one embodiment,the pH value of the aqueous solvent ranges from about 3.5 to about 9.0.

In another embodiment, the aqueous co-solvent comprises DMSO in a rangebetween about 5% and about 10% of the aqueous solution, wherein the pHvalue of the aqueous solution is between about 5.5 and about 7.5. In oneembodiment, the volume ratio of the first phase to the second phase isfrom about 1:9 to about 9:1.

The bioreactor 30 can be a column bioreactor having a solid support thatis impregnated with divalent metal ions or a support whose surface isfunctionalized with divalent metal ions. Typically, sepharose, agarose,or other biopolymers are used as supports for binding divalent metalions such as nickel, cobalt, magnesium, and manganese. Such supportshave a strong affinity for the histidine tag that is present on theexpressed cannabinoid synthase and can be used to sequester the synthaseand separate it from other non-essential proteins and debris that mayinterfere or impede cannabinoid synthesis.

The bioreactor 30 used for synthesizing cannabinoids is configured forbatch and continuous synthetic processes to permit commercial productionof pharmaceutically useful cannabinoids. In one embodiment, thebioreactor 30 is configured for batch synthesis in which the compositionof the medium, concentration of the enzyme and substrate are fixed atthe beginning of the process and not allowed to change during catalysis.Synthesis is terminated when the concentration of the desired product inthe medium of the bioreactor 30 reaches a predetermined value or theconcentration of substrate falls below a predetermined level, such as toa level where there is no detectable catalytic conversion of substrateto product. In one embodiment, therefore, the His-tagged cannabinoidsynthase is sequestered onto a nickel containing resin support withinthe bioreactor column prior to the introduction of a known amount ofsubstrate or cannabinoid precursor, for example, CBGA, CBGVA, or aFormulae II compound into the bioreactor (30). In an alternateembodiment, the cannabinoid precursor is present within the bioreactorhaving a nickel resin support prior to the introduction of the mediumcontaining a cannabinoid synthase into the bioreactor (30). In eithercase, a known amount of the enzyme is contacted with a known amount of acannabinoid precursor to synthesize a cannabinoid or a cannabinoidanalog as product.

In one embodiment, the cannabinoid acid synthase is introduced into thesecond phase within the bioreactor 30 prior to the introduction of aknown amount of substrate of Formula I. The first phase containing thesubstrate of Formula I is then introduced into the bioreactor.

The system, in some embodiments, further includes a filter situatedbetween the fermentor 10 and the bioreactor 30. The filter may filterthe supernatant to at least partially separate the cells from the mediumcontaining the expressed enzyme. Typically, the filter separates atleast 80% of the total cells from the medium. For certain embodiments,the filter separates at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% of the total cells from themedium (4) prior to the introduction of this medium containing thesynthase into the bioreactor. Following filtration, the cells aretransported back to the fermentor, collected for lysate outside thefermentor, or added to the bioreactor. In some embodiments, the filteris a filtration and purification system that includes multiple filtersand reservoirs to purify the cannabinoid synthase.

The progress of the reaction within the bioreactor 30 can be monitoredperiodically or continuously. For instance, an optical monitoring system50 may be utilized to detect the concentration of product in the mediumwithin the bioreactor as a function of time. Alternatively, the decreasein the concentration of substrate can be monitored to signal terminationof synthesis. The cannabinoid product thus produced can be readilyrecovered from the first phase in which the product accumulates. Thecannabinoid or cannabinoids in the first phase are readily purified bysolvent extraction or chromatographic purification methods. Themonitoring system 50 may be part of or may interact with a controlmechanism 40 (a controller) described herein.

An alternative to the batch process mode is the continuous process modein which a defined amount of substrate and medium are continuously addedto the bioreactor (30) while an equal amount of medium containing thecannabinoid product is simultaneously removed from the bioreactor 30 tomaintain a constant rate for formation of product. Medium can enter thebioreactor through an inlet and exit the bioreactor through outlet.Methods of modulating the concentration of substrate, enzyme and otherfactors implicated to maximize the rate of product formation are knownin the art.

An alternative to the batch process mode is another mode in which thefirst phase containing one or more cannabinoid products is removedthough 34 or 35 and the cannabinoid products purified. The first phasecontaining an amount of the substrate (or cannabinoid precursor) ofFormula I is then introduced into the bioreactor through 34 or 35. Theprogress of the reaction is monitored to determine when a sufficientamount of substrate has been converted to product. The removal andreplenishment of the first phase with a predetermined amount ofsubstrate of Formula I can be repeated so long as the cannabinoidsynthase in the second phase remains active.

The conditions of the bioreactor can be controlled using a controlmechanism 40. The control mechanism 40 may be coupled to the bioreactor30 or, alternatively, may interact with the bioreactor 30 wirelessly orremotely. The control mechanism 40 may also be used to control theconditions of the fermentor 10, such as the oxygen level, agitation, pH,pressure, solvent, flow rate, and feed rate. The control mechanism 40may also control the flow of materials (e.g., by controlling at leastone pump) into and out of the fermentor 10 and bioreactor 30. In someembodiments, the control mechanism 40 is configured to control theconditions of at least one of the fermentor 10 and the bioreactor 30based on information obtained from the optical monitoring system 50.

The control mechanism 40 of FIG. 1B may include a processing circuithaving a processor and memory device. The processor can be implementedas a general purpose processor, an application specific integratedcircuit (ASIC), one or more field programmable gate arrays (FPGAs), agroup of processing components, or other suitable electronic processingcomponents. The memory device (e.g., memory, memory unit, storagedevice, etc.) is one or more devices (e.g., RAM, ROM, Flash memory, harddisk storage, etc.) for storing data and/or computer code for completingor facilitating the various processes and functions described in thepresent disclosure, such as controlling the pH, temperature, andpressure of the bioreactor 30, or altering the flow rate of medium intoor out of the bioreactor (30). The processor and memory are configuredto complete or facilitate the various processes and functions describedin the present application, such as controlling the pH, temperature, andpressure of the bioreactor 30, or altering the flow rate of solvents,cells and the like into or out of the bioreactor 30. In someembodiments, for facilitating the control of pH, temperature, pressureand flow rate, the control mechanism 40 may be configured to communicatewith at least one sensor in a sensor suite 60. The sensor suite 60 mayinclude a pH sensor 62, a temperature sensor 63, and a pressure sensor64. The control mechanism 40 may include aproportional-integral-derivative (PID) controller for feedback-basedcontrol. The control mechanism 40 may be further configured to regulatethe flow rate of materials into and out of the fermentor 10 and thebioreactor 30 via pulse width modulation (PWM) techniques. Thebioreactor is able to produce one or more cannabinoids (e.g., a firstcannabinoid and a second cannabinoid) or their analogs. Thus, thecondition of the bioreactor is configured to cause a shift from: 1)formation of the first cannabinoid in greater quantities relative to thesecond cannabinoid to 2) formation of the second cannabinoid in greaterquantities relative to the first cannabinoid. In one embodiment, thecannabinoid so produced from the system comprises (a)tetrahydrocannabivarinic acid (THCVA) and cannabichrome varinic acid(CBCVA), (b) cannabidivarinic acid (CBDVA) and CBCVA, (c)tetrahydrocannabinolic acid (THCA) and cannabichromenic acid (CBCA),and/or (d) cannabidiolic acid (CBDA) and CBCA.

The control mechanism 40 includes a processor 43 coupled to acommunication mechanism 48. The control mechanism 40 further includes amain memory 42, such as a random access memory (RAM) or other dynamicstorage device, coupled to the bus 48 for storing information, andconfigured to store instructions to be executed by the processor 43. Themain memory 42 is further configured to store temporary variables andintermediate information during execution of instructions by theprocessor 43. The control mechanism 40 may additionally include a readonly memory (ROM) 44 or other static storage device connected to the bus48 for storing information and instructions. Additionally, a storagedevice 46, such as a solid state device, magnetic disk or optical disk,may be coupled to the bus 48 for persistently storing information andinstructions.

The present disclosure contemplates methods, systems and programproducts on any machine-readable media for accomplishing variousoperations, such as controlling the conditions of the bioreactor. Theembodiments of the present disclosure may be implemented using existingcomputer processors, or by a special purpose computer processor for anappropriate system, incorporated for this or another purpose, or by ahardwired system. Embodiments within the scope of the present disclosureinclude program products comprising machine-readable media for carryingor having machine-executable instructions or data structures storedthereon. Such machine-readable media can be any available media that canbe accessed by a general purpose or special purpose computer or othermachine with a processor. By way of example, such machine-readable mediacan comprise RAM, ROM, EPROM, EEPROM, CD-ROM, or other optical diskstorage, magnetic disk storage, other magnetic storage devices, solidstate storage devices, or any other medium which can be used to carry orstore desired program code in the form of machine-executableinstructions or data structures and which can be accessed by a generalpurpose or special purpose computer or other machine with a processor.When information is transferred or provided over a network or anothercommunications connection (either hardwired, wireless, or a combinationof hardwired or wireless) to a machine, the machine properly views theconnection as a machine-readable medium. Thus, any such connection isproperly termed a machine-readable medium. Combinations of the above arealso included within the scope of machine-readable media.Machine-executable instructions include, for example, instructions anddata which cause a general purpose computer, special purpose computer,or special purpose processing machines to perform a certain function orgroup of functions.

Furthermore, the control mechanism 40 may be coupled (via the mechanism48) to a display 77, such as a liquid crystal display, or active matrixdisplay, for displaying information to a user. In some embodiments, aninput device 11, such as a keyboard, may also be coupled to the bus 48for communicating information, and to convey commands to the processor43. In some embodiments, the input device 11 has a touch screen display.

The construction and arrangement of the system for producingcannabinoids or cannabinoid analogs as shown in the various exemplaryembodiments are illustrative only. Although only a few embodiments havebeen described in detail in this disclosure, many modifications arepossible (e.g., variations in sizes, dimensions, structures, shapes andproportions of the various elements, values of parameters, use ofmaterials, colors, orientations, etc.) For example, the position ofelements may be reversed or otherwise varied and the nature or number ofdiscrete elements or positions may be altered or varied. Accordingly,all such modifications are intended to be included within the scope ofthe present disclosure. Furthermore, the order or sequence of anyprocess or method steps may be varied or re-sequenced according toalternative embodiments. Other substitutions, modifications, changes,and omissions may be made in the design, operating conditions, andarrangement of the exemplary embodiments without departing from thescope of the present disclosure.

WORKING EXAMPLES Example 1 CBGVA Crystallization in Aqueous Buffer

CBGVA was dissolved at 16 g/L in DMSO and added to 20 mM citrate buffer,pH 4.5 to achieve final CBGVA concentrations in buffer of 0.05, 0.1,0.2, 0.4, and 0.8 g/L and a DMSO concentration of 5% vol/vol. There wasno visible precipitation/crystallization in the 0.05 g/L CBGVA vial, butall of the other vials showed progressively more cloudiness (FIG. 3A).In contrast, vials with CBGA in 20 mM citrate buffer (pH 4.5) and 10%DMSO exhibited precipitation/crystallization, even at 0.05 g/Lconcentration of (FIG. 3B).

These results show that CBGVA is more soluble in aqueous solution thanCBGA. Initially, microscopic examination of the solutions showingcloudiness and/or precipitation revealed small spherical structures.However, after 24 hours large crystals had formed in most of thesolutions (FIG. 4). Both CBGA and CBGVA are more soluble in polarorganic solvents compared to water (data not shown).

Example 2 Biphasic Oil-Aqueous Systems (1:1) Using CBGVA as Substrateand Lyophilized Cannabinoid Synthases (THCA and CBDA Synthases)

Bio-catalysis was performed using a 1:1 biphasic oil:aqueous solventsystem. The oil phase comprising soy-bean oil contained 5 g/L CBGVA. Theaqueous phase comprising citrate buffer, pH 5.5, and 10% DMSO contained32 mg/mL THCA synthase or CBDA synthase. The synthase was previouslylyophilized for storage. A 1:1 ratio (1.5 mL of each) of the aqueous andoil phases were used for bio-catalysis.

The bi-phasic reaction mixture (3 mL total volume) was placed on a tuberotator and agitated at 40 rpm at room temperature. The conversion ofsubstrate to cannabinoid products was monitored by removing samplealiquots of oil at each time point shown in FIGS. 5A-5B. Prior to samplecollection, the vial containing the reaction mixture was removed fromthe tube rotator and the two phases allowed to separate by placing thevial of the reaction mixture on the bench top for about 30 mins. Once aclear separation was visible, 10 μL of oil was diluted in 190 μL ofisopropanol (“IPA”), vortexed, and analyzed by HPLC.

HPLC analysis of the reaction showed that over 90% of the CBGVAsubstrate had been converted by THCA synthase to THCVA and CBCVA after96 hours (FIG. 5A). The ratio of THCVA to CBCVA produced using theoil-water (buffer) solvent system was 4.3:1. After 2 weeks, about 83% ofthe CBGVA had been converted by CBDA synthase to CBDVA and CBCVA, with aCBCVA to CBDVA ratio of 9:1 (minimum amount of THCVA, FIG. 5B). Bothenzymes retained catalytic activity and converted substrate to productover an extended period of 300 hours. Cannabinoid synthesis by the CBDAsynthase also produced THCVA, which was about 2% of total cannabinoidproducts (FIG. 5B).

Example 3 Biphasic Oil-Aqueous Systems (1:1) Using CBGVA as Substrateand Purified CBDA Synthase

This experiment applied the same reaction conditions as Example 2 exceptthat a purified CBDA synthase was used here (instead of a lyophilizedCBDA synthase). Methods to purify CBDA synthase are known in the art. Inthis experiment, the CBDA synthase was purified using an ion exchangechromatography (Sepharose SP Fast Flow resin (GE healthcare lifescience)). In this instance, the experiments were intended to testwhether the activity of the cannabinoid synthase enzyme and/orcannabinoid product profiles differ significantly when a pure enzyme isused for bio-catalysis.

As demonstrated in FIG. 6A, the amount of CBGVA conversion to thecyclized products reached about 88% in a time less than 200 hours. Theratio of CBCVA to CBDVA in this reaction was lower than in thelyophilized enzyme reaction (FIG. 6B). Both lyophilized and purifiedCBDA synthases favored the production of CBCVA.

Example 4 Effects of pH Values on the Production of Cannabinoids fromBiphasic Systems

This purpose of this experiment was to evaluate the effect of pH on theratio of cannabinoid products produced using a biphasic oil-aqueoussolvent system. To observe the effect of pH on reaction kinetics, theamount of enzyme used for bio-catalysis was decreased.

Briefly, 10× stock solutions of THCA synthase and CBDA synthase wereprepared in a solvent comprising 5% DMSO, 95% deionized water. Enzymestocks were diluted 1:10 in five separate tubes containing 100 mM sodiumcitrate buffer at pH values of 4.0, 4.5, 5.0, 5.5, and 6.0 and 5% DMSO.The final enzyme concentration in the aqueous layer comprising 5% DMSOand 95% sodium citrate buffer was about 8.0 mg/mL. 600 μL of soybean oilcontaining 5 mg/mL CBGVA was overlaid onto 600 μL of aqueous phasecontaining crude lyophilized THCA synthase at each of the five pHvalues. Assays were conducted in 2 mL glass HPLC vials and placed on avertical tube rotator at ambient temperature.

Progress of bio-catalysis at each time point was carried using HPLC, bymeasuring the amount of each cannabinoid product synthesized as afunction of time. Briefly, aliquots of oil from each independentreaction vial were collected after removing the vials from the tuberotator and placing them on a bench top for about 30 minutes so as toallow the two solvents of the bi-phasic system to separate. Once clearseparation was visible, 10 μL of oil was pipetted and diluted in 190 μLof IPA, vortexed, and analyzed by HPLC.

The results show that pH does influence cannabinoid product ratios.Based on the pH values examined, the production of THCVA is highest atpH 5.0 (FIGS. 7A-7B). Increasing the pH shifted the product ratio infavor of CBCVA, with the production of CBCVA being substantially greaterat a pH of 6.0 (FIGS. 8A-8B).

pH also influenced cannabinoid product ratios when bio-catalysis iscarried out using the enzyme CBDA synthase. The optimal pH for producingCBDVA using bio-catalysis is pH 5.5 (FIGS. 10A-10B). The amount of CBCVAproduced fell at pH values below the optimal pH of 6.0 (FIGS. 11A-11B).It was interesting to note that while the largest amount of CBDVA wasproduced at pH 5.5, the ratio of CBDVA:CBCVA was at its highest at pH4.5. For CBCVA, the production and the ratio of CBCVA:CBCDA are at theirhighest at pH 6.0. While greater amounts of CBDVA were synthesized at pH5.5 (FIGS. 10A-10B), the synthesis of CBCVA was also greater at pH 5.5than at pH 4.5 (FIGS. 11A-11B). Consequently, in one embodiment,performing bio-catalysis at pH 4.5 using the inventive bi-phasic systemmay be more suitable, as the purification of CBDVA from CBCVA alsoproduced during bio-catalysis would be less time and cost intensive froma mixture that has a larger amount of CBDVA compared to CBCVA,particularly if the desired cannabinoid product is CBDVA.

The CBGVA substrate used for bio-catalysis can be quantified using astandard curve. To generate a standard curve, 10 mg of CBGVA wasdissolved in 10 mL of HPLC grade methanol to a final concentration of1.0 mg/mL. The stock solution was serially diluted 1:1 using the samelot of HPLC grade methanol, resulting in six vials with CBGVA amountsranging from 15.625-500 μg/mL.

The CBGVA substrate can be quantified using a standard curve. Togenerate a standard curve, 10 mg of CBGVA was dissolved in 10 mL of HPLCgrade methanol to a final concentration of 1.0 mg/mL. The stock solutionwas serially diluted 1:1 using the same lot of HPLC grade methanol,resulting in six vials with CBGVA amounts ranging from 15.625-500 μg/mL.

After running a methanol blank, all six samples were analyzed by HPLC at267 nm using an XSELECT CSH Fluoro-Phenyl 3.5 μm 4.6×100 mm column and a57% acetonitrile+0.1% formic acid isocratic method. Purity of CBGVA lotwas determined to be 96.473%. Vial concentrations were adjusted tocorrect for purity and graphed against the absorbance of the CBGVA peak(FIG. 9). The resulting trendline has the formula y=4.958E-05x with anR2 value of 0.9998 (FIG. 9).

Example 5 Effects of DMSO on CBGVA Cyclization by THCA and CBDA Synthasein a Biphasic Oil-Aqueous System

The effect of varying concentrations of DMSO as co-solvent was studiedon the product ratio of cannabinoids produced by bio-catalysis using a1:1 oil-aqueous reaction system. Lyophilized tech-grade THCA synthase orCBDA synthase was dissolved in 100 mM citrate buffer (pH 5.0) containingvarious concentrations of DMSO (1.25%, 2.5%, 5%, 10%, and 20%). Thefinal enzyme concentration in the aqueous buffer was 8.0 mg/mL. 600 μLof soybean oil containing 5 mg/mL CBGVA was overlaid onto 600 μL ofaqueous phase containing either THCA or CBDA synthase at each of theco-solvent concentrations. Assays were conducted in 2 mL glass HPLCvials and placed on a vertical tube rotator at ambient temperature.Reaction progress was monitored by aliquoting a sample of the oil phasefrom each vial at select time points, after allowing the oil phase toseparate from the aqueous phase in each vial. Phase separation isachieved by removing the vials from the tube rotator and allowing eachvial to stand on a bench top for ˜30 minutes. Once clear separation isvisible, 10 μL of oil is diluted in 190 μL of IPA, vortexed, andanalyzed by HPLC.

After 144 hours of reaction, the following trends are visible. THCVAproduction increased as the DMSO concentration increased to 20%, withthe 20% DMSO reactions exhibiting 39.7% conversion after 144 hours (FIG.12A). Also, the ratio of THCVA and CBCVA was its lowest when the DMSOconcentration increased to 20% (FIG. 12B).

CBCVA production was low (less than 3%) for 1.25%-10% DMSO reactions,with the 20% DMSO reaction showing 6.9% CBCVA (FIG. 13A). The ratio ofTHCVA and CBCVA was its highest at 20% DMSO. CBDVA production and theratio of CBDVA to CBCVA were both highest at 1.25% DMSO (FIGS. 14A and14B).

CBDA synthase is more sensitive to DMSO as a co-solvent than THCAsynthase. At 144 hours, CBCVA production was at its highest in 5% and10% DMSO reactions (FIG. 15A) with total CBCVA production at about 12%,although the ratio of CBCVA to CBDVA was better at 10% DMSO (3.42:1)than at 5% DMSO (1.11:1) (FIG. 15B).

Example 6 Biphasic Oil-Aqueous Systems Using CBGVA as Substrate and THCASynthase

In a biphasic oil-aqueous reaction, the oil phase contained 30 g/LCBGVA. The aqueous phase contained 100 g/L technical grade THCAsynthase. The total volume of reaction (including both oil and aqueousphases) was incubated on the tube rotator. At each time point indicatedin FIG. 16, the samples of the oil phase were collected by removing thevials from the tube rotator and allowing the oil and aqueous phases toseparate. Once a clear separation was visible, the oil aliquot wasdiluted in IPA, vortexed, and analyzed by HPLC.

The reaction resulted in a rapid and high conversion (above 95%) ofCBGVA to the varin series of cannabinoid products (THCVA and CBCVA)within about 45 hours since the reaction started. With its highvolumetric efficiency, the biphasic system produced about 24 g/L THCVAand about 5 g/L CBCVA, with a good ratio of THCVA to CBCVA (FIG. 16).

In a separate biphasic oil-aqueous reaction, the oil phase containedabout 14.6 g/L CBGVA as substrate and the aqueous phase contained THCAsynthase. The pH in the aqueous phase was optimized for CBCVAproduction. The total reaction mixture (including both oil and aqueousphases) was incubated on the tube rotator. At each time point indicatedin FIG. 17, the samples of the oil phase were collected by removing thevials from the tube rotator and allowing them to separate. Once a clearseparation was visible, the oil aliquot was diluted in IPA, vortexed,and analyzed by HPLC.

In the reaction, about 70% CBGVA was converted to varin cannabinoidproducts within about 200 hours. (FIG. 17) The reaction produced 11 g/LCBCVA and 2 g/L THCVA with a good ratio of CBCVA to THCVA (around 10:1).

In a similar and separate reaction, more than 99% of the CBGVA wasconverted to varin cannabinoids within 22 hours, and the reactionproduced more than 14 g/L of THCVA and <0.1 g/L THCVA, with a CBCVA toTHCVA ratio of 154:1 (data not shown).

Example 7 Biphasic Oil-Aqueous Systems Using CBGVA as Substrate and CBDASynthase

In a biphasic oil-aqueous reaction, the oil phase contained about 21 g/LCBGVA and the aqueous phase contained CBDA synthase. The total volumereaction (including both oil and aqueous phases) was incubated on thetube rotator. At each time point indicated in FIG. 18, the samples ofthe oil phase were collected by removing the vials from the tube rotatorand allowing them to separate. Once a clear separation was visible, theoil aliquot was diluted in IPA, vortexed, and analyzed by HPLC.

The reaction produced about 10 g/L CBCVA, 7 g/L CBDVA, and about 1 g/LTHCVA with a product ratio favoring CBCVA over CBDVA and THCVA. Thisreaction (FIG. 18) was slightly slower or less efficient than thebio-catalytic reaction optimized for THCVA production (FIG. 16). Thevarin cannabinoid compounds are well resolved by RP-HPLC as shown inFIG. 19.

Example 8 Biphasic Oil-Aqueous Systems (1:1) Using CBGA as Substrate andLyophilized Cannabinoid Synthases (THCA and CBDA Synthases)

In a 1:1 biphasic oil-aqueous reaction, the oil phase contains 5 g/LCBGA dissolved in soybean oil. The aqueous phase contains 32 mg/mL THCAsynthase or CBDA synthase. Both enzymes were reconstituted usinglyophilized enzyme powder and an aqueous buffer. The aqueous phaseincludes citrate buffer and 10% DMSO at pH value at of 5.5. The aqueousphases were combined with oil phases at 1:1 ratio (1.5 mL of each).

The total reaction mixture (3 L) was placed incubated on the tuberotator and spun at 40 rpm at room temperature. At each time pointindicated in FIGS. 20A-20B, the samples of the oil phase were collectedby removing the vials from the tube rotator and allowing them toseparate for about 30 mins. Once a clear separation was visible, 10 μLof oil was diluted in 190 μL of IPA, vortexed, and analyzed by HPLC.

After the same 96 hour period, about 25% of the CBGA was converted byTHCA synthase to THCA and CBCA (FIG. 20A), and about 19% of the CBGA hadbeen converted by CBDA synthase to CBDA and CBCA products (minimumamount of THCA, FIG. 20B). Both reactions were permitted to progress andafter 168 hours, the conversion of CBGA to cannabinoid product reached40% for the THCA synthase reaction with a product ratio of THCA:CBCA of2.2:1 (FIG. 20A). For the CBDA synthase reaction, 30.7% of CBGA wasconverted to cannabinoid products after 2 weeks with a CBCA:CBDA productratio of 1.8:1 (FIG. 20B). As illustrated in FIG. 20B, THCA was producedas a minor product when CBDA synthase is used for bio-catalysis. Theamount of THCA produced is about 0.8% of total cannabinoid products(FIG. 20B).

Example 9 Biphasic Oil-Aqueous Systems (1:1) Using CBGA as Substrate andPurified CBDA Synthase

This experiment applied the same reaction conditions as Example 2 exceptthat a purified CBDA synthase was used (instead of a lyophilizedtechnical grade CBDA synthase). Methods to purify CBDA synthase areknown in the art. In this experiment, the CBDA synthase was purifiedusing ion exchange chromatography and Sepharose SP Fast Flow resin (GEhealthcare life science).

As illustrated by the graph in FIG. 21A, about 20% of the CBGA substrateis converted to cannabinoid products after 300 hours. Compared to crudelyophilized CBDA synthase, the purified CBDA synthase produced higheramounts of CBDA product with a CBDA:CBCA ratio of 2.1:1 after 2 weeks ofreaction (FIG. 21B). The amount of CBDA produced after 2 weeks was 509.8mg/L (data not shown). Purified enzyme may affect the cannabinoidproduct ratios.

Example 10 Effects of pH Values on the Production of Cannabinoids fromBiphasic Systems

This experiment evaluated the effect of pH (e.g., pH above 6.0) on thecannabinoid production in a biphasic oil-aqueous system. In order tomore accurately observe the effect of pH on reaction kinetics, theamounts of loaded enzymes were lowered.

10× stock solutions were prepared for THCA synthase and CBDA Synthase in5% DMSO, 95% deionized water. Enzyme stocks were diluted 1:10 using fivebuffers containing 100 mM sodium citrate and 5% DMSO at pH values of4.0, 4.5, 5.0, 5.5, and 6.0. The final enzyme concentration in theaqueous layer was about 8.0 mg/mL in 5% DMSO and 95% sodium citratebuffer at variable pHs. 600 μL of soybean oil containing 5 mg/mL CBGAwas overlaid onto 300 μL of aqueous phase containing crude lyophilizedTHCA synthase at each of the five pH values. Assays were conducted in 2mL glass HPLC vials and placed on a vertical tube rotator at ambienttemperature.

For each time point, samples of the oil phase were collected by removingthe vials from the tube rotator and allowing them to separate for ˜30mins. Once clear separation was visible, 10 μL of oil was diluted in 190μL of IPA, vortexed, and analyzed by HPLC.

For each time point, samples of the oil phase were collected by removingthe vials from the tube rotator. The results showed that among all ofthe pH values examined, the production of THCA reached its greatest atpH 5.5 (FIG. 22A), while the production of CBCA was the highest at pH7.5 (FIG. 23A).

Example 11 Effects of Various Oil to Aqueous Phase Ratios on theProduction of Cannabinoids in Biphasic Aqueous-Oil Systems

This experiment was designed to investigate the effect of varying oil toaqueous ratios while keeping the absolute amount of CBGA and enzyme inthe system constant. In this experiment, stock solutions of CBGA andlyophilized THCA synthase were prepared as shown in Table 1. The CBGAand THCA synthase solutions in oil and aqueous buffer were combined inthe ratios listed in Table 1 using five separate 1 mL reaction vials.Each entry in Table 1 is carried out in duplicate (vial set A and vialset B).

TABLE 1 Experimental Conditions of oil-aqueous ratio assays CBGA EnzymeVial Concentration Volume Total Concentration Volume Total # (mg/mL)(mL) (mg) (mg/mL) (mL) (mg) 1 60.61 0.33 20 60.61 0.66 40 2 50.00 0.4 2066.67 0.6 40 3 40.00 0.5 20 80.00 0.5 40 4 33.33 0.6 20 100.00 0.4 40 530.30 0.66 20 121.21 0.33 40

All vials in set A were incubated at ambient temperature on a tuberotator spinning at 40 rpm and removed 30 minutes prior to sampling. A10 μL sample was taken from the oil phase of each vial at each timepoint and combined with 190 μL of IPA, vortexed, and injected on theHPLC for analysis.

On the other hand, the vials in set B were allowed to incubate asdescribed above, without sampling in the middle of reaction. At the endpoint for set B, which was determined by monitoring the reactions in setA, the vials in set B were extracted (total reaction extraction with 9volumes of IPA). One 10 μL sample of oil phase was extracted prior tothe total reaction extraction.

The ratio of oil to aqueous phase influenced the amount of cannabinoidproducts produced as well as the ratio of cannabinoid products producedusing the biphasic system. As shown in FIG. 24, a biphasic reactionmixture comprising 33% oil and 66% aqueous buffer (1:2 oil-aqueousratio) produced the maximum amount of total cannabinoid product (FIG.24). Ratios of THCA:BCA products at each time point are shown in FIG.25A, while the ratios of THCA:CBCA products at 408 hours are shown inFIG. 25B.

For the vials in set B, the oil extract and total reaction extractsproduced nearly identical percentage of CBGA and products (FIG. 26).

Efficiency of bio-catalysis, at least based on mass balances was good.The slight difference between theoretical yields and actual yields maybe due to errors introduced during the removal and transfer of solventsand reaction mixture. FIG. 27 illustrates the amounts of CBGA, THCA, andCBCA as well as total cannabinoid produced for each oil:aqueous bufferratio. It is evident from this figure that the calculated sum of THCA,CBCA and CBGA for each oil to aqueous buffer ratio are not significantlydifferent from the total cannabinoid content (blue bar) estimated usingthe total reaction extract (vial B).

Example 12 Activity of Purified THCA Synthase in (1:1) BiphasicOil-Aqueous Systems with Varying pHs and DMSO Concentrations

This experiment is designed to determine the effects of DMSO on thebioconversion of CBGA to THCA and/or CBCA at pH 5.5 and pH 7.5 using apurified enzyme preparation.

In the first experiment, the aqueous phase contains 0.1 M citratebuffer, 270 μg/mL purified THCA synthase with 5%, 10%, or 20% DMSO at pH5.5. The soybean oil phase contained 20 g/L CBGA. FIG. 28A shows thetotal amount of cannabinoid product that accumulates over time. FIG. 28Bshows the amounts of THCA, CBCA, and the combination of THCA and CBCAthat is produced for each concentration of DMSO after 672 hours.

In the second experiment, the aqueous phase contains 0.1 M HEPES bufferand 270 μg/mL purified THCA synthase with 5%, 10%, or 20% DMSO at pH7.5. The soybean oil phase contains 20 g/L CBGA. At pH 7.5, theconversion continues to progress 500 hours after initiation ofbiocatalysis. The greatest amount of CBCA was produced in the reactionwith 10% DMSO (FIG. 29).

Example 13 Activity Purified THCA Synthase in Biphasic Oil-AqueousSystems (1:1) with Lower Amounts of DMSO Co-Solvent or in the Presenceof Methanol Concentrations as Co-Solvent

This experiment was designed to evaluate the effect of low amounts ofDMSO or methanol on biocatalysis using CBGA and purified THCA synthase.The 1:1 biphasic oil-aqueous reactions contained 272m/mL purified THCAsynthase in 100 mM citrate buffer and various amounts of DMSO ormethanol at pH 5.5. The oil phase contains 10 g/L CBGA.

As shown in FIG. 30, the reaction containing 10% methanol produced thehighest amount of total cannabinoids, although this condition producedthe lowest ratio of THCA to CBCA (FIG. 31A and FIG. 31B).

Also, the reaction solutions with 5% and 2.5% DMSO produced amounts oftotal cannabinoids that are comparable to the 10% methanol reactionsolution (FIGS. 13A-13B), but with much higher ratio of THCA to CBCA atabout 2.5:1, compared with 0.7:1 for the 10% methanol reaction solution(FIG. 31A and FIG. 31B).

Example 14

Reaction of CBGA cyclization catalyzed by CBDA synthase was studied inaqueous solutions over a broad range of reaction times andconcentrations of organic co-solvents, methanol and DMSO (from 1 to 20%,v/v). The reactions were stopped by quenching with equal volume of MeOHafter 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 5 hours, and 23hours and the reaction products were analyzed on LC-MS/MS. A typicalUV-HPLC trace showing major reaction products is presented in FIG. 32.

The reaction solution contained 0.1 M citrate buffer, 20 mg/mL CBDAsynthase, 0.2 mg/mL CBGA at pH 4.5, and with different volumepercentages of DMSO or methanol (MeOH). Here, CBGA was introduced intothe reaction from a 100-fold dilution of stock solution in MeOH (20mg/mL) that introduced 1% (v/v) MeOH into the reaction. The 1 mLreaction solutions in glass vials were shaken at 75 rpm in PrecisionScientific thermostat water bath at 25° C.

After different incubation time, aliquots of each reaction (0.1 mL) werediluted 2-fold by mixing with 0.1 mL MeOH; centrifuged at 4° C. at 9,990rpm and supernatant was injected in LCMS for the product determination.FIG. 32 shows the UV-HPLC trace of products in the presence of 10% (v/v)MeOH after two hours.

As shown in FIGS. 33A-33B, generation of CBDA follows linear kineticsunder all solvent conditions for at least first 30 minutes of thereaction. Kinetics of CBDA formation is not significantly influenced bythe presence of methanol up to 15% (v/v). Addition of 20% (v/v) methanolproduces some suppression of the reaction as shown in the lower curve ofFIG. 33A. Increasing concentrations of DMSO significantly decreases therate of CBDA accumulation (FIG. 33B).

Also, THCA was produced as a minor product in the reaction catalyzed byCBDA synthase in comparison with CBDA (FIGS. 34A-34B). An increase inthe concentration of methanol results in increased production of THCA(FIG. 34A). Conversely, an increase in the concentration of DMSO resultsin decreased production of THCA (FIG. 34B).

The rapid production of CBCA is shown in FIGS. 35A and 35B. ComparingFIG. 35B to FIG. 35A, DMSO was more efficient in catalyzing theproduction of CBCA with higher percentages of co-solvents DMSO andmethanol.

Example 15 THCA Synthase Activity after Lyophilization

First, THCA synthase from an IEX resin was lyophilized and stored at−20° C. One month later, one vial of the lyophilized enzyme was removedfrom the freezer, warmed to room temperature, and dissolved andreconstituted in 1 mL of deionized water. 0.9 mL of reconstituted enzymesolution was added to 0.1 mL of 1.0 mg/mL CBGA stock in DMSO, mixedbriefly, and placed on a tube rotator at ambient temperature. Theactivity of enzyme was monitored over 2 hours with samples taken at 15,30, 60, and 120 minutes. Samples (100 μL) were extracted with an equalvolume of methanol, centrifuged, and analyzed by HPLC.

As shown in FIG. 36, the lyophilized THCA synthase retains its catalyticactivity after one year of storage.

Example 16 Stability of CBDA Synthase in the Presence of PolarCo-Solvents

The effects of varying concentrations of co-solvents (methanol or DMSO)on the stability of CBDA synthase were evaluated. Here, 20 mg/mL CBDAsynthase was incubated in 100 mM citrate buffer (pH 4.5) in the presenceof different volume percentages of polar co-solvents (MeOH and DMSO).The solutions were shaken at 75 rpm in Precision Scientific thermostatedwater bath at 25° C. After different time intervals, CBGA was introducedby mixing aliquot of 100-fold concentrated stock solution of CBGA inMeOH (20 mg/mL) with aliquot of incubated enzyme. After incubation for30 minutes, the reactions were stopped by mixing with an equal volume ofMeOH (2-fold dilution), centrifuged at 4° C. at 9990 rpm and supernatantwas injected in LCMS for determination.

Changes in the activity of CBDA synthase after incubation (for up to 23hours) at pH 4.5, 25° C., with different concentrations of MeOH (up to10%, v/v) and DMSO (up to 20%, v/v) are shown in FIGS. 37A and 37B,respectively.

Incubation with methanol, prior to measuring enzyme activity of CBDAsynthase, did not produce significant effect on the activity of theenzyme (FIG. 37A). In contrast, the addition of DMSO prior to activitymeasurements significantly reduced the activity of CBDA synthase, andthe effect was more pronounced at higher DMSO concentrations. Incubationwith 20% (v/v) DMSO resulted in almost complete loss of the enzymeactivity. Inactivation of CBDA synthase by DMSO was rapid, as after thefirst hour of incubation the enzyme activity loss was significant anddid not change much after the following incubation with DMSO up to 23hours (FIG. 37B). A similar result was observed for THCA synthase (datanot shown).

Example 17 Biphasic Oil-Aqueous Systems Using CBGA as Substrate and THCASynthase

In a biphasic oil-aqueous reaction, the oil phase contained 36 g/L CBGA.The aqueous phase contained THCA synthase. The pH in the aqueous phasewas optimized for CBCA production. The total volume reaction (includingboth oil and aqueous phases) was incubated on the tube rotator. At eachtime point indicated in FIG. 38, the samples of the oil phase werecollected by removing the vials from the tube rotator and allowing themto separate. Once a clear separation was visible, the oil aliquot wasdiluted in IPA, vortexed, and analyzed by HPLC.

The reaction resulted in a rapid, high conversion (above 95%) of CBGA tothe cannabinoid products (THCA and CBCA) within about 160 hours sincethe reaction started. With its high volumetric efficiency, the biphasicsystem produced about 30 g/L of cannabinoid products (about 29 g/L CBCAand about 2 g/L THCA) with the excellent product ratio of CBCA to THCA(>40:1) in the final products (FIG. 38).

In a separate biphasic oil-aqueous reaction, the oil phase contained 44g/L CBGA as substrate and the aqueous phase contained THCA synthase. ThepH in the aqueous phase was optimized for THCA production. The totalvolume reaction (including both oil and aqueous phases) were incubatedon the tube rotator. At each time point indicated in FIG. 39, thesamples of the oil phase were collected by removing the vials from thetube rotator and allowing them to separate. Once a clear separation wasvisible, the oil aliquot was diluted in IPA, vortexed, and analyzed byHPLC. This reaction also resulted in a rapid, high conversion (above90%) of CBGA to the cannabinoid products (THCA and CBCA) within about160 hours, and produced more than 30 g/L of cannabinoid products (about19 g/L THCA and about 13 g/L CBCA) (FIG. 39). The large amount ofcannabinoid products suggested a high volumetric efficiency of thisbiphasic reaction.

Example 18 Biphasic Oil-Aqueous Systems Using CBGA as Substrate and CBDASynthase

In a biphasic oil-aqueous reaction, the oil phase contained 20 g/L CBGAsubstrate and the aqueous phase contained CBDA synthase. The pH in theaqueous phase was optimized for CBDA production. The total volumereaction (including both oil and aqueous phases) was incubated on thetube rotator. At each time point indicated in FIG. 40, the samples ofthe oil phase were collected by removing the vials from the tube rotatorand allowing them to separate. Once a clear separation was visible, theoil aliquot was diluted in IPA, vortexed, and analyzed by HPLC.

The reaction with CBGA substrate (FIG. 40) was still efficient inproducing cannabinoid products, although it is slightly slower and has alower volumetric efficiency than the above reactions with THCA synthase(FIG. 38). At 140 hours post-reaction, about 50% of CBGA substrate wasconverted to cannabinoid products (>9 g/L), among which the CBDA productwas about 6 g/L and the CBCA product was about 2 g/L (FIG. 40). Thecannabinoid compounds were well resolved by RP-HPLC as shown in FIG. 41.

Example 19 Biphasic Oil-Aqueous Systems in a Scale-Up Reaction

The bioconversion reaction of CBGVA to THCVA and CBCVA with THCAsynthase was performed in a scale-up reactor (a 3 L stirred-tankreactor). Example 2 showed an efficient conversion of CBGVA to THCVA andCBCVA using tech-grade lyophilized THCA synthase in a small scalereaction (3 mL total volume) (FIG. 5A). This scale-up reaction wasconducted to demonstrate the ability to conduct the reaction on 30 g ofcannabinoid substrate using THCA synthase enzyme at 100 g/L.

CBGVA Solution in Oil Phase

1 L of soybean oil and 35 g of CBGVA were mixed in substrate in a 2 Lbottle on an orbital shaker at 37° C. and 120 rpm over two days, afterwhich the CBGVA solution in soybean oil appeared hazy and exhibited somebrown insoluble clumps at the bottom and brown insoluble materialadhered to the glass of the bottle. The solution was centrifuged in aSorvall RCSC floor centrifuge at 9,000 rpm for 10 minutes to clarify thesolution.

The CBGVA solution was analyzed by a HPLC machine, which estimated theconcentration to be 26 g/L. Additional solution with 7 g of CBGVA wassolubilized in 200 mL of soybean oil after overnight shake andcentrifuge and was added to the initial CBGVA solution to make the batchconcentration. The combined CBGVA solution had an estimatedconcentration at 28 g/L based on the HPLC analysis.

3 L Reaction System

The 3 L bioreactor components were assembled with agitation, pH control,and temperature control. The reactor vessel was placed on a support ringattached to a scaffold and secured with two large chain clamps. Rushtonimpellers were secured to the Teflon-coated stir shaft, with the lowerimpeller 3 cm above the bottom of the shaft, and the top impellerpositioned so that it was just above the 2 L mark on the reactor. Thetop of the stir shaft was passed through the center opening of the headplate and secured in a variable-speed stirring mechanism attached to thesupport scaffold. The headplate was clamped in position with thequick-release clamp. The reactor jacket inlet and outlet were attachedto the temperature control unit.

A glass addition funnel was secured to the support scaffold with twolarge chain clamps, size 16 Pharmed tubing was attached to the stopcock,and the tubing was run through a Watson-Marlow 120U/DV benchtopperistaltic pump. This pump was connected to the pH controller and setto a deadband of +/−0.05 pH units. 2 N HCl was added to the funnel andthe line was primed. The pH probe was connected to the pHmonitor/controller and calibrated using both pH 7 and pH 4 buffers.

THCA Synthase in Aqueous Phase

2 L of 100 mM sodium citrate buffer (pH 5.0) and 10% DMSO (v/v) wereprepared as follows:

-   -   1. Dissolving 13.45 g of anhydrous citric acid (Fisher A940-1)        in 700 mL of deionized water to make a 100 mM solution;    -   2. Dissolving 38.23 g of sodium citrate dihydrate (Sigma        W302600-1KG-K) in 1.3 L of deionized water to make a 100 mM        solution;    -   3. Mixing the citric acid and sodium citrate solutions, removing        200 mL solution, and adding 200 mL of DMSO (Sigma 276855-1L);        and    -   4. Mixing DMSO within the solution and adjusting pH to 5.0 with        2 N HCl.

200 g of tech-grade THCA synthase (BPD1090-F500) was added with 1.6 L ofcitrate buffer into a 5 L bucket. The solution with THCA synthase wasmixed with a spatula.

Bioconversion Reaction

The aqueous solution with the THCA synthase (100 g/L THCA synthase in100 mM sodium citrate buffer with 10% DMSO and pH 5.0) was introduced tothe 3 L bioreactor through the headplate using a funnel with the startstirring speed at 250 rpm. The remaining 400 mL of citrate buffer wasused to rinse the bucket and added into the reactor. A pH probe wasinserted and clamped into position. The enzyme solution was warmed to37° C. An activated pH control pump brought the pH to the bottom of thedeadband (pH 4.95). 1.1 L of CBGVA substrate in soybean oil was added tothe reactor using a long-stemmed glass funnel. All unused ports werecapped. Parafilm was applied around pH probe and stir shaft to minimizeevaporation.

The bioconversion reaction was monitored with the following samplingprocedure:

-   -   Pausing the mixing process to allow the reactor to sit for one        minute;    -   Sampling the upper phase via a serological pipette (˜1 mL        removed) and resuming the mixing process;    -   Centrifuging the sample at 20,800 rpm in a 1.5 mL tube for 5        minutes to facilitate clear separation of oil and aqueous        phases;    -   Sampling the oil phase, diluting the sample in 1:50 with IPA,        and analyzing the sample by HPLC as shown in table 2.

TABLE 2 HPLC analysis XSELECT CSH Fluoro-Phenyl 3.5 μm 4.6 × 100 mmColumn 10 μL injection volume Solvent A: Water with 0.1% Formic AcidSolvent B: Acetonitrile with 0.1% Formic Acid Time (minutes) Solvent B %0 57 12 57 13 95 15 95 16 57 17 57 Flow rate = 1.0 mL/min, Temperature =30° C.

The conversion of CBGVA to THCVA and CBCVA progressed at a steady rateover a period of 55 hours, after which conversion levelled off. FIG. 41shows the conversion of CBGVA to THCVA and CBCVA, as measured bypercentage of HPLC peak area at 267 nm. At 92 hours, once it wasverified that the reaction was completed, the reactor was harvested andextracted. Nearly all of the CBGVA substrate was converted, with <2%remaining based on the HPLC peak area. In the products, THCVA accountedfor 77% of the HPLC peak area at the end of the reaction; CBCVAaccounted for 18% of the HPLC peak area at the end of the reaction. Therate of conversion of CBGVA levelled off significantly after 54.5 hours,with ˜5% remaining. The reaction could have been harvested at thispoint, but it was allowed to progress to completion to minimizeinterference from CBGVA in downstream purification (and to determine themaximum level of conversion achievable).

In addition to tracking cannabinoids based upon the percentage of HPLCpeak area, absolute amounts of cannabinoids were calculated fromstandard curves to confirm mass balance (FIG. 42). The standard curveprovides a more accurate estimate of produced cannabinoids than a methodbased on the percentage of HPLC peak area because the HPLC percent areachart examines only one wavelength.

Harvest and Extraction

When the reaction agitator stopped, the reaction solution slowlyseparated to three distinctive phases, which were removed by drainingthrough the bottom outlet valve (BOV).

TABLE 3 Estimated quantities of cannabinoids in each reaction phase asmeasured by HPLC % peak area, mg/mL concentration, and total grams (thelatter two based on standard curves) Vol. CBGVA THCVA CBCVA Sample mL %Area mg/mL g % Area mg/mL g % Area mg/mL g Lower 1050 0.78 0.002 0.00262.20 0.15 0.16 14.47 0.02 0.25 Aqueous Middle 800 1.40 0.04 0.031 74.332.25 1.80 18.07 0.37 0.29 Aqueous Upper 1350 1.48 0.32 0.43 76.65 18.2924.69 18.02 2.89 3.91 Emulsion

As shown in Table 3, the lower aqueous phase contained very minoramounts of cannabinoids; the middle phase contained more cannabinoids,but still a minor fraction of the total; the majority of thecannabinoids were found to be present in the upper oil emulsion phase,as expected. The oil emulsion was transferred to a 6 L separatory funneland extracted with 4 L of methanol by mixing vigorously for 1 minute andallowing the vessel to rest. 3.5 L of methanol phase was recovered andfiltered.

About 500 mL of methanol stayed with the oily phase as an emulsion andwas not collected until later. The methanol extraction was repeatedthree more times using 3.5 L of methanol and 1.8 L of upper oilyemulsion phase. Each time the extract was filtered by gravity throughWhatman 2v folded filter paper, which clarified the solutions. As thesequential extractions were performed, the 1.3 L of oily phase began toseparate more from the ˜500 mL MeOH emulsion phase. 400 mL of emulsionphase was collected, filtered, and analyzed. Phase contained only tracecannabinoids was discarded. All four methanol extracts were pooled andmethanol was removed via evaporation under reduced pressure in a 20 Lrotovap at 30-35° C., which yielded about 100 mL of viscous orange oil.The oily material was resuspended using 200 mL of DI water.

The aqueous/oil solution was also extracted with 3.0 L of heptanes bymixing vigorously in a 4 L separatory funnel and allowing the phases toseparate. The phases separated more rapidly than methanol extractions ofthe oil phase, which resulted in thick emulsions. The heptanes extractwas vacuum filtered using a Whatman 3 filter to remove particulates.FIG. 43 showed the amount of THCVA extracted from each stage based onthe HPLC analysis.

Since the varin cannabinoids are more polar than the standardcannabinoids, a second extraction was performed using 1 L of 9:1heptanes-ethyl acetate. HPLC analysis showed that this was unnecessary,as the extract contained only trace amounts of cannabinoids. MinimalMgSO₄ was added to the heptanes extract to remove water. The solutionwas stirred and filtered by gravity using a fluted Whatman filter,resulting in a clarified solution. The heptanes solution was evaporatedunder reduced pressure. Due to the cannabinoids' ability to entrainheptanes, the material was resuspended in methanol and again subjectedto evaporation under reduced pressure followed by vacuum dryingovernight at room temperature.

The final crude extract was weighed at 52.76 g, which contained anestimated 22.38 g of THCVA and 3.58 g of CBCVA, with about 50%gravimetric purity. The mass balance of THCVA through the extractionprocess is summarized in FIG. 43.

Silica Column Chromatography.

A small-scale (15 g/35 mL) silica column purification was conducted on asmall side sample of the extract to guide the planned larger (2.5 kg/5L) columns. 370 mg of dried cannabinoid extract from the 3 L scale upwas dissolved in 2 mL of heptanes. 1 mL solution containing 185 mg crudeextract with about 90 mg total cannabinoid was loaded on 15 g of silicaequilibrated with heptanes with a bed volume of 35 mL. The flow rate wasset to 1.0 mL/minute and the column was treated with 3 CV of 95:5heptanes-ethyl acetate. The first two fractions were collected as asingle bed volume (BV) (35 mL volume) while subsequent fractions werecollected as 0.5 BV (17.5 mL). Solvent was changed to 9:1 heptanes-EAafter 3.0 CV and again changed to 8:2 heptanes-EA after a total of 7.0BV before finally switching to 1:1 to facilitate rapid CBCVA elutionafter 11 BV.

The elution profile is consistent with expectations. The THCVA appearedin trace amounts after three bed volume equivalents with the majority ofthe material eluting between 4.0 and 5.0 CV (FIG. 44). The fractiondirectly preceding THCVA elution was dried down and found to contain 38mg of gravimetric impurity. Fractions containing greater than 99% pureTHCVA were pooled and found to contain 53.29 mg with a gravimetric massof 81.90 mg. This pooled material had a gravimetric purity of 65% and anHPLC purity of 99.74%.

A large-scale silica column was used to process half of the crudecannabinoid extract from the 3 L reactions converting 30 g of CBGVA toTHCVA and CBCVA. In the large-scale elusion, solvent was changed to 9:1heptanes-EA after 4.0 CV and again changed to 8:2 heptanes-EA after atotal of 8.0 BV before finally switching to 1:1 to facilitate rapidCBCVA elution after 10 BV. Fractions were sampled (100 μL) and driedunder nitrogen and dissolved in 200 μL of methanol for HPLC analysis.The elution profile is consistent with the observed elution profile atsmall-scale (data not shown). In the large-scale elusion profile,fractions 6-32 (CV 3.09-7.82) contained >98% pure THCVA by HPLC at 267nm and were combined and dried under reduced pressure to yield anestimated 9.76 g of THCVA with an HPLC purity of 99.2%. About 89% of theTHCVA was loaded onto the column. The final weight of the dried THCVApool was 12.49 g with a 78% gravimetric purity. The remaining fractionswas divided into various pools and dried for storage and potentialisolation at a later time.

THCA Synthase Stability and Potential for Recycle

Given the linear conversion of CBGVA to THCVA and CBCVA over the courseof the 3 L bioconversion reaction (FIG. 45), the enzymatic activity wasevaluated immediately following harvest of the reaction at 92 hours. Arecycle experiment was performed with the small-scale stationaryreaction. In the experiment, only the oil layer was replaced, leavingthe aqueous layer and a thin interface layer intact. Significantactivity of THCA synthase was retained in the small scale experiment.The reaction using recycled enzyme proceeded at approximately a third ofthe rate of the original reaction (FIG. 46).

Recycle of the aqueous and interface layer was investigated by usingvarious permutations of soybean oil/dipentene and tech grade/enrichedenzyme. Each tech grade enzyme aqueous phase was prepared at 100 mg/mL.Each enriched enzyme aqueous phase was prepared at 1 mg/mL. Organicphases contained 30 g/L CBGVA. For each reaction, 500 uL organic phasewas added to 1 mL aqueous phase. After the reaction was initiated, theorganic phase was removed and replaced with organic phase containingfresh substrate every 18-24 hours. As shown in FIGS. 47A-47B and48A-48B, significant enzymatic activities were retained with tech-gradeenzyme through 3 reaction cycles. Enriched enzymes exhibited similarinitial activity, but lost activity much faster. In the soybean oil withtech-grade enzyme reaction, overall production increased to 32.0 mgTHCVA over 4 recycles from 7.6 mg THCVA in a single reaction (FIG. 49).

Example 20 Terpene as the Organic Solvent of the Biphasic System

Reaction with THCA Synthase

Addition of catalase in the aqueous phase or dipentene in the organicphase had a beneficial impact on the efficiency of cannabinoid enzyme(data not shown). Here, Applicant tested if simultaneous addition ofcatalase and dipentene can further reduce the volume of enzyme in theconversion reaction. Tech-grade THCA synthase was dissolved to 100 g/Lin 100 mM sodium citrate at pH 5. The stock solution was without 10%DMSO to avoid potential adverse effects on catalase in the aqueousphase. This stock solution was used to make aqueous phases at 25 g/L, 33g/L, and 50 g/L THCA synthase. Catalase was added to each diluted THCAsynthase solution to a final concentration of 0.1 mg/mL. Aqueous phasesat each enzyme concentration were layered with dipentene containing 30g/L CBGVA to initiate the reaction. The reactions were placed in a 37°C. incubator at 40 rpm and sampled over several days. The extent ofreaction (as measured by consumption of substrate) is shown in FIG. 51A.

The 50 g/L enzyme reaction achieved full conversion within 20 hours.Surprisingly, the 33 g/L reaction also achieved almost full conversionin about 48 hours. The reaction with 25 g/L THCA synthase did notachieve completion. 33 g/L THCA was comparably lower than previousexperiment to fully convert 30 g/L CBGVA. For example, the concentrationwas one third of that used in the 3 L scale-up demonstration (100 g/L)in Example 19.

A separate experiment was conducted to test if 25 g/L THCA synthase canfully convert 30 g/L CBGVA through co-solvent optimization. Here,tech-grade THCA synthase was dissolved to 25 g/L in 100 mM sodiumcitrate (pH 5) with 0.1 mg/mL catalase and 0%, 5%, 10%, or 15% DMSO.Aqueous phases with DMSO were layered with dipentene containing 30 g/LCBGVA to initiate the reaction. The reactions were placed in a 37° C.incubator at 40 rpm and sampled over two days. The extent of reaction(as measured by consumption of substrate) is shown in FIGS. 50A and 50B.As previous experiment, 25 g/L enzyme with no co-solvent did not achievecomplete conversion of CBGVA. However, the conversion of CBGVA wascompleted with 5%, 10%, and 15% DMSO. Increasing the concentration ofDMSO in aqueous solution also shifted the product ratio toward CBCVA(FIG. 50C). At a high DMSO concentration (15%), the overall productionof THCVA decreased. Without being bound by a theory, a moderateconcentration of DMSO (5-10%) favors the production of THCVA withoutsignificant amounts of CBCVA.

To further test the effect of dipentene on CBGVA conversion, a similarexperiment was carried in a scale-up system (300 mL). 3 g of CBGVA wasdissolved in 100 mL of dipentene in a 250 mL bottle (30 g/L), withincubation at 37° C. and intermittent swirling. After several minutes,the CBGVA appeared to have fully dissolved, with a brownish layer ofmaterial on the bottom of the bottle. The CBGVA concentration wasestimated at 31 g/L with HPLC analysis. After overnight incubation, theCBGVA solution was centrifuged at 6,800×g for 5 minutes to remove theformed crystals and then was measured at 29 g/L.

In setting up the 300 mL jacketed reactor, Applicant took extra measuresto minimize losses of organic phase to evaporation, including adding astir shaft collar, replacing the headplate gasket with a type thatprovides a better seal, and using plugs and parafilm to block otherpotential points for evaporative loss. The temperature control unit wasset at 37° C. and the reactor jacket was circulated with solution. TwoRushton impellors were aligned with the bottom impellor placed as closeas possible to the bottom of reactor. The top impellor was placed ataround 200 mL mark on reactor. The pH probe was calibrated with pH 7 andpH 4 standards.

5 g of lyophilized tech-grade THCA synthase was dissolved in 200 mL 100mM sodium citrate buffer with 10% DMSO (pH 5.0) and 20 mg of catalase.The enzyme solution was added to the reactor and was stirred at 250 rpm.The pH probe was inserted and secured on the reactor with pH maintainedat 5.0. The gaps around the probe were sealed with parafilm. Once thetemperature of enzyme solution stabilized at about 36° C., 100 mL ofclarified dipentene solution containing CBGVA was added to the reactor.

As shown in FIG. 50D, the reaction progressed rapidly to completionwithin 20 hours. Particularly, over 11 g/L of THCVA were produced after4 hours, and ratios of THCVA to CBCVA remained at around 7:1 throughoutthe reaction. After 20 hours the reaction was complete, with only 0.3mg/mL of CBGVA substrate remaining in the dipentene, and 28.86 g/L ofTHCVA produced. Over the course of the reaction, the total cannabinoidconcentration increased gradually from 29 g/L to 33 g/L, suggesting thatthere was some minimal loss of dipentene.

As noted above, replacing soybean oil with dipentene in the organicphase can increase the efficiency of biphasic reactions at a low pH.Here, the production of CBCVA was further evaluated at a higher pH withdipentene and various cosolvents. Four different aqueous buffers weremade using different cosolvents with the buffer adjusted to pH 7 afterthe cosolvent was added:

-   -   i. Condition 1: 100 mM HEPES, pH 7, 30 mg/mL THCA synthase        BPD1090-500, and 0.1 mg/mL Catalase (no cosolvent)    -   ii. Condition 2: 100 mM HEPES, pH 7, 30 mg/mL THCA synthase        BPD1090-500, 0.1 mg/mL Catalase, and 10% DMSO    -   iii. Condition 3: 100 mM HEPES, pH 7, 30 mg/mL THCA synthase        BPD1090-500, 0.1 mg/mL Catalase, and 10% MeOH    -   iv. Condition 4: 100 mM HEPES, pH 7, 30 mg/mL THCA synthase        BPD1090-500, 0.1 mg/mL Catalase, and 20% DMSO

400 μL of dipentene solution containing about 30 mg/mL CBGVA wasoverlaid onto 800 μL of the aqueous buffer. In addition, 400 μL ofsoybean oil containing comparable CBGVA was overlaid onto another vialcontaining aqueous condition 4 to act as a control. The reactionsolutions were sampled at various time points over two days and aresummarized in FIG. 50E. The addition of a co-solvent had a positiveeffect on the production of CBCVA when compared to the non-co-solventcontrol.

The standard soybean oil with 20% DMSO reaction significantlyoutperformed all of the reactions with dipentene and had the highestratio of CBCVA to THCVA of any reaction (FIG. 50F). Very little THCVA(less than 5%) was produced in these reactions. Without being bound by atheory, it appears that dipentene had little effect on the reactionsthat occur at higher pH.

CBDA Synthase Reaction

CBDA synthase was dissolved to 100 mg/mL in 100 mM sodium citrate, pH 5.1 mL of this enzyme solution was added to 500 uL of 30 g/L CBGVA ineither dipentene or soybean oil. The reaction was then incubated at 37°C. on a 40 rpm rotator and sampled over several days. The dipentenereaction performed significantly better than the soybean oil reaction(FIG. 51A), producing over twice the amount of CBDVA. Interestingly,unlike THCA synthase, the initial rates were similar between dipenteneand soybean oil, and the faster rate was sustained longer in dipentene.

To further investigate the effects of co-solvent and catalase on CBDAsynthase, 100 mg/mL 900 uL CBDA synthase (100 mg/mL) solution (100 mMsodium citrate, pH 5) was mixed with different co-solvents (100 uLadditional buffer, 100 uL MeOH, or 100 uL 1 mg/mL catalase). Then, 500uL of 30 g/L CBGVA in either dipentene or soybean oil was added to theCBDA synthase solution to initiate the reaction. The reaction was thenincubated at 37° C. on a 40 rpm rotator and sampled over several days.

Biosynthesis with CBDA synthase using soybean oil is less efficient thandipentene. Compare FIG. 51B with FIG. 51C. Catalase appeared to allowthe dipentene reaction to sustain at a high conversion rate for a longerperiod of time (FIG. 51B). This beneficial effect seems absent, however,in the soybean oil reaction (FIG. 51C). This is consistent withApplicant's previous observation that catalase can promote thehigh-productive reactions.

The inclusion of methanol as a co-solvent enhanced production of totalcannabinoids (FIG. 51D).

To further determine the optimal concentration of methanol for the CBDAsynthase activity, 40 mg/mL CBDA synthase in five buffers (100 mM sodiumcitrate, pH 5) with different concentrations of methanol (0%, 1%, 2.5%,5%, or 10%). 400 μL of dipentene or soybean oil each containing about 10mg/mL CBGVA was overlaid onto 800 μL of aqueous enzyme solution.Reactions took place in 2.0 mL glass vials and were placed on a verticaltube roller (30 rpm) at ambient temperature. Samples were taken over sixdays to assess the production of CBDVA. The dipentene-MeOH biphasicsystem produced more total cannabinoids (CBDVA, THCVA, & CBCVA) than thesoybean oil-MeOH system (FIGS. 51E and 51F). Further, 10% MeOH improvedthe total cannabinoid production with either soybean oil or dipentene(FIGS. 51E and 51F).

Comparison of Dipentene and Soybean Oil Reactions

To further compare the biphasic reactions with dipentene and soybeanoil, standard 1.5 mL reactions were set up using 100 g/L THCA synthaseand 10% DMSO in the aqueous phase and 30 g/L CBGVA in either dipenteneor soybean oil as the organic phase. At each time point, both the oilphase and aqueous phase were sampled, using an exhaustive samplingprocedure to minimize any organic contamination of the aqueous samples.As illustrated in FIG. 52, cannabinoid substrate concentrations aregreater in dipentene than in soybean oil. The mass transfer ofcannabinoid substrate from dipentene into the aqueous phase was morerapid than the mass transfer of cannabinoid substrate from soybean oilinto the aqueous phase (FIG. 52).

EQUIVALENTS

It is to be understood that while the disclosure has been described inconjunction with the above embodiments, the foregoing description andexamples are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages, and modifications within thescope of the disclosure will be apparent to those skilled in the art towhich the disclosure pertains.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

The embodiments illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation, orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by specific embodiments and optionalfeatures, modification, improvement, and variation of the embodimentstherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements, and variations are consideredto be within the scope of this disclosure. The materials, methods, andexamples provided here are representative of particular embodiments, areexemplary, and are not intended as limitations on the scope of thedisclosure.

The scope of the disclosure has been described broadly and genericallyherein. Each of the narrower species and subgeneric groupings fallingwithin the generic disclosure also form part of the disclosure. Thisincludes the generic description with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatembodiments of the disclosure may also thereby be described in terms ofany individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

1-84. (canceled)
 85. A system for producing a cannabinoid or its analog,comprising: (a) fermenter holding a medium and a plurality of cells,wherein the cells are configured to produce and secrete cannabinoidsynthase; (b) a bioreactor containing a composition comprising acannabinoid precursor in a first phase and a cannabinoid synthase in asecond phase; and (c) a control mechanism configured to control at leastone condition of the bioreactor to modify the amount of a firstcannabinoid relative to the amount of a second cannabinoid or itsanalog, wherein the control mechanism comprises a processing circuithaving a processor and a memory device; wherein the first phasecomprises an organic solvent and the second phase comprises an aqueoussolvent.
 86. The system of claim 85, wherein the control mechanism isconfigured to control at least one of pH, solvent, temperature,pressure, and flow rate of the composition in the bioreactor to modifythe amount of the first cannabinoid relative to the amount of the secondcannabinoid or its analog.
 87. The system of claim 85, wherein the firstphase of the bioreactor is agitated to form micro-droplets within thesecond phase, wherein at least one micro-droplet comprises thecannabinoid precursor.
 88. The system of claim 87, wherein the firstphase is agitated to form micro-droplets within the second phase by animpeller in the bioreactor.
 89. The system of claim 85, wherein thecannabinoid synthase is introduced to the bioreactor through a headplateusing a funnel.
 90. The system of claim 85, further comprising: (a) afilter configured to at least partially separate the plurality of cellsfrom the medium, such that after separation the medium containscannabinoid synthase produced by the plurality of cells; and/or (b) asensor suite configured to facilitate the control of the condition bythe control mechanism, wherein the sensor suite comprises one or more ofa pH sensor, a temperature sensor, and a pressure sensor.
 91. The systemof claim 85, wherein the bioreactor is a column bioreactor containingnickel, and wherein the cannabinoid synthase includes a tag configuredto bond to nickel.
 92. The system of claim 85, wherein a change in thecondition of the bioreactor is configured to cause a shift from: 1)formation of the first cannabinoid in greater quantities relative to thesecond cannabinoid to 2) formation of the second cannabinoid in greaterquantities relative to the first cannabinoid.
 93. The system of claim85, wherein the cannabinoid precursor is cannabigerolic acid (CBGA),cannabigerovarinic acid (CBGVA), or their derivative or analog.
 94. Thesystem of claim 85, wherein the cannabinoid precursor is the compound ofFormula II:

wherein R₁ is H or —COOH and R₂ is a linear or branched CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₀, C₆H₁₃, C₇H₁₅ or C₈H₁₇ group, wherein the cannabinoidprecursor is configured to interact with the cannabinoid synthase toform the cannabinoids or its analog.
 95. The system of claim 85, whereinthe first phase is substantially water-immiscible or water-immiscible.96. The system of claim 85, wherein the organic solvent comprises one ormore of olive oil, sesame oil, castor oil, cotton-seed oil, soybean oil,butane, pentane, heptane, octane, isooctane, nonane, decane, andterpene.
 97. The system of claim 96, wherein the terpene comprises: (a)one or more of hemiterpene, monoterpene, sesquiterpene, diterpene,sesterterpene, triterpene, sesquarterpene, tetraterpene, polyterpene,and norisoprenoid, or (b) one or more of diterpene, tri-terpene,myrcene, β-caryophyllene, limonene (or dipentene), pinene, and linalool.98. The system of claim 85, wherein the aqueous solvent furthercomprises one or more of dimethyl sulfoxide (DMSO), dimethylformamide(DMF), dimethylacetamide (DMA), isopropyl alcohol, cyclodextrin,peroxide scavenger, and methanol (MeOH).
 99. The system of claim 98,wherein the amount of the aqueous solvent is between about 0.001% andabout 50% (w/v), about 1% and about 40%, about 1% and about 30%, orabout 1% and about 20% of the second phase.
 100. The system of claim 98,wherein the aqueous solvent comprises the peroxide scavenger in anamount between about 0.001% and about 0.1%, about 0.005% and about0.05%, or about 0.01% and about 0.03% of the aqueous solution.
 101. Thesystem of claim 98, wherein the peroxide scavenger is one or more ofcatalase, glutathione peroxidases (GPx), thioredoxin-assistedperoxidases (Prx), sodium pyruvate, and N,N′-dimethylthiourea (DMTU).102. The system of claim 101, wherein the aqueous solvent comprisescatalase in an amount between about 0.001% and about 0.1%, about 0.005%and about 0.05%, or about 0.01% and about 0.03% of the aqueous solution.103. The system of claim 85, wherein the aqueous solvent comprises: (a)DMSO in an amount between about 0.1% and about 50% of the aqueoussolution; (b) DMSO in a range between about 5% and about 10% of theaqueous solution, wherein the pH value of the aqueous solution isbetween about 5.5 and about 7.5; or (c) MeOH in an amount between about1% and about 20% of the aqueous solution.
 104. The system of claim 85,wherein: (a) the pH value of the aqueous solvent ranges from about 3.5to about 9.0; (b) the volume ratio of the first phase to the secondphase is from about 1:9 to about 9:1; (c) the cannabinoid synthasecomprises CBDA synthase or THCA synthase; and/or (d) the concentrationof cannabinoid precursor in the bioreactor is between about 0.1 mg/mLand about 250 mg/mL.
 105. The system of claim 85, wherein the volumeratio of the first phase to the second phase is about 1:2.
 106. Thesystem of claim 85, wherein the volume ratio of the first phase to thesecond phase is about 2:1.
 107. The system of claim 85, wherein thecannabinoid produced comprises: (a) tetrahydrocannabivarinic acid(THCVA) and cannabichrome varinic acid (CBCVA), (b) cannabidivarinicacid (CBDVA) and CBCVA, (c) tetrahydrocannabinolic acid (THCA) andcannabichromenic acid (CBCA), or (d) cannabidiolic acid (CBDA) and CBCA.108. A system for producing a cannabinoid or its analog, comprising: a)a bioreactor containing a composition comprising a cannabinoid precursorin a first phase and a cannabinoid synthase in a second phase, whereinthe bioreactor is configured to agitate the first phase to formmicro-droplets within the second phase, and b) a control mechanismconfigured to control at least one condition of the bioreactor to modifythe amount of a first cannabinoid relative to the amount of a secondcannabinoid or its analog, wherein the control mechanism comprises aprocessing circuit having a processor and a memory device, wherein thefirst phase comprises an organic solvent and the second phase comprisesan aqueous solvent.
 109. The system of claim 108, wherein the firstphase is agitated to form micro-droplets with the second phase byrotating, vibrating, vortexing, swirling, shaking, ultrasonicating, orstirring.
 110. The system of claim 109, wherein the first phase isagitated to form micro-droplets within the second phase by an impellerin the bioreactor or by a rotator.
 111. The system of claim 108,wherein: (a) the cannabinoid precursor is CBGA, CBGVA, or theirderivative or analog; (b) the cannabinoid synthase comprises CBDAsynthase or THCA synthase; (c) the organic solvent comprises one or moreof olive oil, sesame oil, castor oil, cotton-seed oil, soybean oil,butane, pentane, heptane, octane, isooctane, nonane, decane, andterpene; and/or (d) the volume ratio of the first phase to the secondphase is from about 1:9 to about 9:1.
 112. The system of claim 108,wherein the volume ratio of the first phase to the second phase is about1:2.
 113. The system of claim 108, wherein the volume ratio of the firstphase to the second phase is about 2:1.
 114. The system of claim 108,wherein the cannabinoid produced comprises (a) THCVA and CBCVA, (b)CBDVA and CBCVA, (c) THCA and CBCA, or (d) CBDA and CBCA.
 115. Abiphasic composition comprising: (a) a cannabinoid precursor in a firstphase, wherein the cannabinoid precursor is the compound of Formula II:

wherein R₁ is H or —COOH and R₂ is a linear or branched CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₀, C₆H₁₃, C₇H₁₅ or C₈H₁₇ group; and (b) a cannabinoid synthasein a second phase, wherein the first phase and the second phase aresubstantially immiscible or immiscible.
 116. A method for producing acannabinoid product comprising: contacting a cannabinoid precursor in afirst phase with a cannabinoid synthase in a second phase, wherein thecannabinoid precursor is the compound of Formula II:

wherein R₁ is H or —COOH and R₂ is a linear or branched CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₀, C₆H₁₃, C₇H₁₅ or C₈H₁₇ group, wherein the first phase andthe second phase are substantially immiscible or immiscible.